[Establishment of STO Cell Lines Expressing Green Fluorescent Protein and Mouse Leukemia Inhibitory Factor].

OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION: The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.

Economic burden of epilepsy in a developing country: a retrospective cost analysis in China.

PURPOSE: To study the cost of epilepsy in China, and, therefore, provide essential information on the burden of the disease to individuals and society. METHODS: A cost-of-illness study was performed on a retrospective cohort of medically treated patients. Data on clinical characteristics, utilization of sources, and costs were collected from 289 patients in a standardized format. RESULTS: Direct medical care costs was Chinese yuan, renminbi (RMB) 2,529 (USD 372) per year per patient, of which antiepileptic drugs (RMB 1,651 or USD 243) accounted for the major cost component. Nonmedical direct costs were much less than direct health care costs, averaging approximately RMB 756 (USD 111). Costs due to loss of productivity averaged approximately RMB 1,968 (USD 289) per patient per year. Taken together, the overall mean annual cost for epilepsy per patient in our series was approximately RMB 5,253 (USD 773), and these costs accounted for more than half of the mean annual income. Total cost was significantly associated with disease severity and different responses to drug treatment. In addition, new antiepileptic drugs and the number of drugs taken were closely related with the drug cost. CONCLUSION: The results indicate that the economic burden of epilepsy to both Chinese patients and the nation is heavy, and the composition proportions of the costs in China have many similar features and some noteworthy differences with that of other countries.

[Construction of recombinant adenovirus vector for RNA interference with multidrug resistance MDR1 gene].

OBJECTIVE: To construct the recombinant adenoviral RNA interference (RNAi) vector in order to inhibit the expression of multidrug resistance MDR1 gene, and probe whether gene therapy for multidrug resistance of epilepsy is feasibility. METHODS: Three target sequences for short hairpin RNA (shRNA) expression were selected and designed according to MDR1 gene sequence of rat. Annealed oligos were inserted into the downstream of treated pSIREN-shuttle U6 promoter to construct RNAi plasmid pSIREN-shuttle-MDR1. Next, MDR1 shRNA sequence was cloned to pAdeno-X, a transfer vector of adenovirus, to produce the pAdeno-MDR1, which was then packed and amplified in HEK293 cells. Further the recombinant adenovirus was purified by CsCl and used to infect the rat astrocytes with P170-glucoprotein (P-gp) over-expression which have been induced by coriaria lactone (CL). RESULTS: It was confirmed by restriction digestion, PCR and sequencing that MDR1 shRNA expression structure was correctly cloned to pSIREN-shuttle and pAdeno-X vector respectively. Virus titer was 6 x 10(9) pfu/mL. The interference efficiency of pAdeno-MDR1 to the expression drop of multidrug resistance gene in astrocyte model neared to 100%. CONCLUSION: RNAi adenovirus vector of rat MDR1 gene has been constructed and found its high interference efficiency. It is the essential building required for the remedy of refractory epilepsy and the research on mechanism of multidrug resistance.