ABSTRACT
Objective To study the condition and method of hydrodynamics-based transgene(HDT) in rat fatty liver. Methods Inject different dosages and concentrations of green fluorescent protein plasmid pEGFP-C1 at different speeds, then collect 4 rats' liver leaves at different time points after injection and prepare their frozen section, finally observe and quantify the GFP expression with fluo rescence microscope at 488 nm excitation wavelength. Results Plasmid pEGFP-C1 concentration 33mg/L, injection speed 2ml/s, injection volume 8.5%of rat body weight, injected plasmid. After 6 hours of injection, GFP-positive cells rate of pedicel leaf is about 18%, left leaf about 14%, middle leaf about 12.5%;R3ight leaf about 10% and tail leaf about 8%. GFP begin to gradually reduce since 24 hours, until 72 hours almost no GFP-positive cells were checked in all liver leaves. Conclusion Hydrodynamics-based transgene can be applied to rat fatty liver, the appropriate conditions of this method are 33mg/L plasmid concentration, 8.5% rat avoirdupois, 2ml/s injection speed, and the suitable time to observe the proportion of GFP-positive cells is 6-24 hours after gene injection.
ABSTRACT
Objective To study the conditions and methods of hydrodynamics-based transgene into rat regenerating liver in vivo. Methods The solution with concentration 30mg/L gene-containing plasmid was injected into rat tail veins at a speed of 2ml/s, then partial hepatectomy (PH) was performed at different times before/after injection, finally the rat (g) and regenerating liver (g) were weighed, and the liver coefficient (Lc) was calculated. Out of 15 groups which are Lc±Lc*0%, *5%, *10%, *15%, *20%, *25%, *30%, *35%, the most suitable group was chosen as correction coefficient to calculate the most appropriate volume of plasmid solution which was injected into the regenerating liver at different recovery times, and at the same time, right lobe of liver was gathered to make frozen section, then observe and quantify the positive green fluorescent protein (GFP) rate at 488 nm excitation wavelength. Results Injection of either physiological saline or empty plasmid has no significant difference compared with control (only PH performance). The appropriate time of hydrodynamics-based transgene is more than 12 hours before PH or anytime after PH. The solution volume of hydrodynamics-based transgene into liver regenerating rat after PH is rat weight (g) ×9%×1/3×corresponding correction coefficient (Trc). Both vector and target gene have effect on the time and abundance of gene expression. Conclusion Hydrodynamics-based transgene can effectively be applied to gene transfection in rat regenerating liver.
ABSTRACT
Objective To investigate aberrant methylation in the promoter area of p16 gene and p16 protein expression in human pancreatic carcinoma and in the corresponding tumor-adjacent tissues,and evaluate their role in the carcinogenesis and progression of tumor and its clinical significance.Methods Immunohistochemistry and MSP(methylation-specific PCR)were performed on 46 samples of pancreatic carcinoma and their corresponding tumor-adjacent tissue specimens for p16 and its methylation.Results Expression rate of p16 protein was 41.3%(19/46)in pancreatic carcinomas,95.7%(44/46)in corresponding tumor-adiucent tissues.Through MSP,the methylation rate in pancreatic carcinomas was 39.1%.No gene methylation was found in 19 cases expressing p16 protein.p16 gene methylation was closely related to p16 protein expression in pancreatic carcinoma(P0.05);but were significantly related to the PTNM staging,histological differentiation,distant metastasis and lymph node metastasis(P