| ObjectiveTo determine the method of constructing the bioartificial renal tubule assist device (RAD) in vitro and the effect of RAD treating MODS/MOF with ARF animal models. Further investingation was taken to optimize the function of RAD by gene transfection. It was expected that RAD could simulate the physiological function of renal tubule in vitro and innovate the technique of renal replacement therapy nowadays, which only replace the filtration function of gromeruli, to increase the survival rate of patients.MethodsThe cell culture of LLC-PK1 was performed. Then the cells were seeded into the lumen of the hemofilter. The filter was kept on incubating while the activity of cells was detected by MTS. Then the filtration in vitro was simulated to detect the reabsorption rate of sodium, creatinine, glucose and four kinds of amino acids. The effect of oubaine and phlorizin on the reabsorption rate of sodium, glucose and amino acids was also observed as compared with no LLC-PK1 device.Pigs which undergone cecal ligation and puncture (CLP) plus bilateral ureteral ligation were randomly divided into four groups after the diagnosis ofMODS/MOF and ARF was established. They are group A which was treated with RAD, group B which was treated with sham RAD containing no cells, group C which was received traditional CVVH and group D without treatment. Blood biochemical markers including electrolytes, urea nitrogen and creatinine, serum cytokines (IL-10, TNF-) and survival time were recorded or measured.Ad5E1A gene was amplified by RT-PCR from 293 cell line. Then the production of RT-PCR was cut by the restriction enzyme KpnI and Ehol. The enzymed vector pShuttle CMV and insert were ligated with T4-ligase. Recombinated pShuttle CMV -ad5ElA was transfected the primary cultured human proximal tubule cells (PTC) and the positive clone was identifyied by RT-PCR and immunofluorescence for ad5E1A mRNA and protein expression. The growth property of transfected cells was determined by MTT and FACS method. The CKP, LDH, NAG and Na + -K+-ATPase level of transfected cells was detected for the functional dertermination as compared with HK2 cell line.ResultsThe creatinine reabsorpion rate of RAD was significantly decreased as compared with control group (P<0.01). At first, the reabsorption rate of sodium, glucose and amino acids had no difference between two groups (P>0.05). Then the reabsorption rate of sodium, glucose and amino acids of RAD group was significantly inhibited by oubaine and phlorizin (P<0.01) and can be recovered after oubaine andphlorizin were drived out (P>0.05).As to the animal model research, there had no difference of MAP between four groups before treatment. The MAP of RAD group (group A) rose significantly after treatment as compared with sham RAD group (group B), regular CVVH group (group C) and non-treatment group (group D) (P<0.01). The serum creatinine (Scr) of four groups had no difference before treatment. After 24 hours of treatment, the Scr of group A and C decreased obviously as compared with group B (P<0.05). The Scr of group A had no difference with group C. The peak level of IL-10 of group A was significantly higher than other three groups (P<0.01). The serum TNF-a level of group A dropped obviously after 24 hours of treatment and had significant discrepancy as compared with that before the treatment and group C at the same time point (P<0.05). The TNF-a concentration of both group B and D had no statistically significant change during the treatment. The average survival time of group A was significantly longer than other three groups (P<0.05).During the research of construction of immortalized tubule cells, ad5E1A gene was amplified from 293 cell line by RT-PCR correctly. After the ad5E1A gene was cut by double restriction enzyme KpnI and EhoI and ligated with pShuttle CMV vector, the recombinated plasmid was transfected the PTC with lipofectamine. The positive clone could be selected out and cultured more than 30 passages stably in vitro. It had been documented that ad5E1A gene and...
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