Study On The Roles Of The Activable Cell-Penetrating Peptides(ACPP) Marked With FITC And Gd-DTPA In Intrahepatic Bile Duct Epithelial Cells Of Human

Objective:To study the changes of human intrahepatic bile duct epithelial cells of cholepathy,the paramagnetic gadolinium or fluorescein was dragged in cell by the utilization of cell-penetrating peptides, and then the feasibility of supervising the changes of intrahepatic bile duct epithelial cells of cholepathy in vitro and drug targeting was discussed. This study will provide new therapy for cholepathy and improve the curative effects of cholepathy.Methods : 1. Synthesis and label of cell-penetrating peptides: activatble cell-penetrating peptide(EEEEEEEE–Ahx-PLGLAG-RRRRRRRR-Ahx-k) was synthesised by Shanghai Top-peptide Biological technology Co.,Ltd.by solid phase method,labeled with fluorescein(FITC) or paramagnetic gadolinium(Gd-DTPA) alone at the N end. 2. Fluorescence microscope detection:the normal intrahepatic bile duct epithelial cells of human was cultured, after training to the logarithmic growth phase,the cell were inoculated in 6 orifice plate exposing to 2μg/ml、5μg/ml、10μg/ml、15μg/ml、20μg/ml of LPS respectively for 24 h, and then incubated with 25umol?L-1、50umol?L-1、100umol?L-1、150umol?L-1 of ACPP labeled with FITC(FITC-ACPP) for 2h, the fluorescence expression in all of the cell above and the cell which exposed to LPS for 24h, incubated with FITC but no ACPP for 2h and the cell which incubated with FITC-ACPP for 2h but not exposed to LPS were observed.Choose the best concentration according to the experimental results. 3. Flow cytometry analysis:(1)h IBDEC were cultured in culture flask exposing to 2.5μg/ml、5μg/ml、10μg/ml of LPS respectively for 72 h,and then incubated with 100umol?L-1 of FITC-ACPP for 2h.(2) the h IBDEC were exposed to5μg/ml of LPS for 24h、48h、72h respectively, and then incubated with 100umol?L-1 of FITC-ACPP for 4h.(3) the h IBDEC were exposed to 5μg/ml of LPS for 72 h, and then incubated with 100umol?L-1 of FITC-ACPP for 1h、2h、4h respectively. The fluorescence expression in all of the cell above were observed with flow cytometry. 4. Magnetic Resonance Imaging research:(1)HIBDEC were cultured in culture flask exposing to 2.5μg/ml、5μg/ml、10μg/ml of LPS respectively for 72 h, and then incubated with 100umol?L-1 of ACPP labeled with Gd-DTPA(Gd-DTPA- ACPP)for 2h.(2)the h IBDEC were exposed to5μg/ml of LPS for 24h、48h、72h respectively, and then incubated with 100umol?L-1 of Gd-DTPA- ACPP for 4h.(3)the h IBDEC were exposed to 5μg/ml of LPS for 72 h, and then incubated with 100umol?L-1 of Gd-DTPA- ACPP for 1h、2h、4h respectively. The average signal intensity in all of the cell above and the cell which incubated with Gd-DTPA- ACPP for 4h but not exposed to LPS and the cell which not exposed to LPS,not incubated with Gd-DTPA- ACPP were observed with MRI. visualization analysis also be used to observe the variation tendency of all the cell. Results: 1. Fluorescence microscope detection show that there is no fluorescence expression in the cell which exposed to LPS for 24 h, incubated with FITC but no ACPP for 2h and the cell which incubated with FITC-ACPP for 2h but not exposed to LPS, all of the rest can be seen the expression of fluorescence.Comparing the fluorescence expression of the cell which incubated with same concentration of FITC-ACPP but diffirent concentration of LPS, there was no obvious diffirent between these groups, for the groups of 15μg/ml and 20μg/ml,because the concentration was too high for the cell to survive,there is no research results of them. Comparing the fluorescence expression of the cell which exposed to same concentration of LPS but diffirent concentration of FITC-ACPP, the groups of 25umol?L-1 and 50umol?L-1were lower than the groups of 100umol?L-1and 200umol?L-1. Between the groups of 100umol?L-1 and 200umol?L-1,the fluorescence expression has no significant difference,so choose the 100umol?L-1 of FITC-ACPP as the experiment concentration. All of the fluorescence expression above were judged by naked eye. 2. Flow cytometry analysis suggest that(1)The intracellular fluorescence intensity of h IBDEC were significantly increased with the incubation time of FITC-ACPP extend from 1h to 4h, undering the condition of which the h IBDEC were exposed to 5μg/ml of LPS for 72 h, and then incubated with 100umol?L-1 of FITC-ACPP,(P < 0.05).(2) The intracellular fluorescence intensity of h IBDEC were significantly increased with the stimulation time of LPS extend from 24 h to 72 h, undering the condition of which the h IBDEC were exposed to5μg/ml of LPS, and then incubated with 100umol?L-1 of FITC-ACPP for 4h,(P < 0.05).(3) The intracellular fluorescence intensity of h IBDEC were different among the groups of different stimulating concentration of LPS,but there] is not significantly difference(P > 0.05). 3. Magnetic Resonance Imaging research show that(1) The average signal intensity of h IBDEC were significantly increased with the incubation time of Gd-DTPA-ACPP extend from 1h to 4h, undering the condition of which the h IBDEC were exposed to 5μg/ml of LPS for 72 h, and then incubated with 100umol?L-1 of Gd-DTPA-ACPP,(P < 0.05), the phenomenon also valid by visualization analysis.(2) The average signal intensity of h IBDEC were significantly increased with the stimulation time of LPS extend from 24 h to 72 h, undering the condition of which the h IBDEC were exposed to5μg/ml of LPS, and then incubated with 100umol?L-1 of Gd-DTPA-ACPP for 4h,(P < 0.05), the phenomenon also valid by visualization analysis.(3) The average signal intensity of h IBDEC were different among the groups of different stimulating concentration of LPS,but there is not significantly difference(P > 0.05), the phenomenon also valid by visualization analysis. The average signal intensity of all of the cell above were higher than the cells which incubated with Gd-DTPA- ACPP for 4h but not exposed to LPS and the cell which not exposed to LPS,not incubated with Gd-DTPA- ACPP,(P < 0.05). Conclusion: ACPP marked with FITC and Gd-DTPA has obvious targeting and efficienttransmembrane activity. The transmembrane activity is related to the incubation time of ACPP and the stimulation time of LPS,with which can be used to trace the status of h IBDEC irritated by LPS.