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| 1 | Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method显示文摘AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro. METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of NucleofectorTM electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectincoated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination.The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4+ ES implantation. RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION: Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, thepotential of pax-4-nucleofected cells in medical treatment is promising. | Han-Tso Lin Chung-Lan Kao Kun-Hsiung Lee Yuh-Lih Chang Shih-Hwa Chiou Fu-Ting Tsai Tung-Hu Tsai Dey-Chyi Sheu Larry LT Ho Hung-Hai Ku | 2007 | World Journal of Gastroenterology2007，13，11： | 10 |
| 2 | Preoperative assessment of corneal and refractive stability in soft contact lens wearing photorefractive candidates显示文摘 | Ng LT Lee EM Nguyen AL | 2007 | Optom Vis Sci2007，84，5： | 1 |
| 3 | Optimal cut-off values for obesity:using simple anthropometric indices to predict cardiovascular risk factors in Taiwan显示文摘 | Lee LT Chen CY | 2002 | Int J Obes Relat Metab Disord2002，26，： | 1 |
| 4 | Blockade of the epidermal growth factor receptor ty rosine kinase activity by quercetin and luteolin leads to growth inhibition and apo ptosis of pancreatic tumor cells 显示文摘 | Lee LT Huan YT Hwan JJ | 2002 | Anticancer Res2002，22，3： | 1 |
| 5 | Cholangitis in children with biliary atresia: health-care resource utilisation 显示文摘 | Lee JY Lim LT Quak SH | 2014 | J Paediatr Child Health2014，50，3： | 1 |
| 6 | Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group 显示文摘 | Wang LT Lee FL Tai CJ Kasai H | 2007 | Int J Syst Evol Micr2007，57，： | 1 |
| 7 | Effects of personal particulate matter on peak expiratory flow rate of asthmatic children显示文摘 | Tang CS Chang LT Lee HC | 2007 | Sci Total Environ2007，382，： | 1 |
| 8 | Diagnostic sub-types, psychological distress and psychosocial dysfunction in southern Chinese with temporomandibular disorders 显示文摘 | Lee LT Yeung RW Wong MC | 2008 | J Oral Rehabil2008，35，3： | 1 |
| 9 | Low plasma nitrite in in-fantile hypertrophic pyloric stenosis patients显示文摘 | Huang LT Tiao MM Lee SY | 2006 | Dig Dis Sci2006，51，5： | 1 |
| 10 | Opposite association between diabetes, dyslipidemia, and hepatocellular carcinoma mortality in the middle-aged and elderly显示文摘 | Chiang CH Lee LT Hung SH | 2014 | Hepatology2014，59，6： | 1 |
| 11 | Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteolin leads to growth inhibition and apoptosis of pancreatic tumor cells显示文摘 | Lee LT Huang YT Hwang JJ | 2002 | Anticancer Res2002，22，3： | 1 |
| 12 | A case of trichloroethylene hypersensitivity syndrome 显示文摘 | Goon AT Lee LT Tay YK | 2001 | Arch Dermatol2001，137，3： | 1 |
| 13 | Cholangitis in children with biliary atresia: Health-care resource utilisation 显示文摘 | Lee JY Lira LT Quak SH | 2014 | J Paediatr Child Heahh2014，50，3： | 1 |
| 14 | A case of trichloroethylene hypersensitivity syndrome显示文摘 | Coon AT Lee LT Tay YK | 2001 | Arch Dermatol2001，137，3： | 1 |
| 15 | The Thin Plate as a Prior in Bayesian SPECT Reconstruction 显示文摘 | Lee SJ Hsiao LT Gindi G | 1997 | IEEE Trans Nucl Sci1997，44，3： | 1 |
| 16 | Detection of Listeria mono2 cytogenes and the toxin listeriolysin 0 in food显示文摘 | Robin LT Churchill Hung Lee | 2006 | Journal of Mi 2 crobiological Methods2006，64，： | 1 |
| 17 | Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteoin leads to growth inhibilion and apoptosis of pancreatic tumor cells 显示文摘 | Lee LT Huang YT Hwang JJ | 2002 | Anticancer Res2002，22，3： | 1 |
| 18 | Novel application of artificial dermis plus autologous vital epithelial cells: improved wound epithelialization显示文摘 | Lee LT Kwan PC Wong YK | 2010 | J Chin Med Assoc2010，73，： | 1 |
| 19 | Optimal cut off values for obesi- ty: using simple anthropometric indices to predict cardiovascular risk factors in Taiwan显示文摘 | Lin WY Lee LT Chen CY | 2002 | lnt J Obes Relat Metab Disord2002，26，9： | 1 |
| 20 | Metabolic syndrome is related to nonalcoholic steatohepatitis in severely obese subjects显示文摘 | Lin WY Lee LT | 2007 | Obes Surg2007，17，： | 1 |
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