Study On Expression Of FasL In Rat Liver Through High Volume Hydrodynamic Injection Of Naked DNA Via The Portal Vein

Objective:To develop the experimental method of high volume hydrodynamic injection of naked plasmid DNA with report gene via the rat portal vein,which can result in report gene expression in rat liver.To construct the eukaryotic vector conbinant with rat FasL.To observe the overexpression of FasL in hepatic tissue, especially in hepatocyte,and the correlation between liver injury,biochemical indicator alteration of liver function and FasL expression.To investigate whether hepatocytes which are simulated to over express FasL in vivo can become effector cells which induce hepatocyte to apoptosis or necrosis.Methods:(1)Naked plasmid DNA of pDC316-LacZ was injected to rat liver in high volume hydrodynamic way via portal vein.Under temporal clamping of inferior vena cava with haemostat,the portal vein was punctured with a scalp acupuncture connected with a syringe,then 400μg naked plasmid of pDC316-LacZ solution in 5 ml of 5%glucose was injected at a rate of 1ml/s.The temporal occlusion was not unclamped until 5 minutes after injection.We harvested the rat liver after 24 hours to detect the expression of LacZ gene.(2)Rat FasL cDNA was cloned and inserted into eukaryotic vectors of pcDNA3.1/HisA and pDC315.The two plasmids were transfected into HepG2 cells respectively. The cells were harvested after 48 hours to identify expression capability of the plasmids by RT-PCR and Western blot.(3)Eleven Wister rats were divided into test group which was treated with pDC315-rFasL plasmid and control group which was treated with pDC316-LacZ through the method mentioned above.Two rats were sacrificed each time on day 1,day3/4,day6 after surgery.Rat sera were harvested before and after surgery in order to be assayed for aminopherase.The livers were harvested for detecting expression of FasL in rat liver by RT-PCR, Western blots and immunohistochemistry,and for observing effect to hepatic tissue after overexpression of FasL in rat liver by HE staining.Rusult:(1)The expression ofβgalactosidase was found in rat liver which was injected with pDC316-LacZ plasmid,implying successful establishment of gene tansfer method by high volume hydrodynamic injection of naked DNA via portal vein.(2)The construction of pcDNA3.1/HisA-rFasL and pDC315-rFasL had been achieved,and expressions of two vectors in HepG2 cell had been verified by RT-PCR and Western blot.(3)RT-PCR and Western blot on hepatic tissues harvested at the first day after FasL gene delivery proved that FasL had been expressed in rat liver successfuly.Immunohistochemistry results indicated that FasL had expressed abundantly in the hepatic lobule first region of rats liver in test group,but not in control group.The expression of FasL decreased gradually and reached a very low level at day 6.The HE staining showed that the hepatocytes in hepatic lobule first region of test group appeared more acidophilia degeneration and apoptosis than thoese of control group at day 1,but oncotic necrosis of hepatocyte and infiltration of inflammatory cell had not emergenced. These changes recovered gradually,and returned to nomal at day 6.The serum transaminase increased significantly at day 1 in both groups.The transaminase value in control group recovered to normal level at day 3/4,while maintaining abnormal level in test group,which recovered to normal at day 6.Conclusion:(1)high volume hydrodynamic injection of naked plasmid DNA via the hepatic portal vein have been initially developed,which provide a relative safe and effective method for gene delivery.(2)Two eukaryotic vectors of pcDNA3.1/HisA-rFasL and pDC315-rFasL have been constructed and transfected to HepG2 cell in vitro successfully.Thirdly,delivering the naked plasmid with FasL to rat liver displayed that FasL expressed beyond nomal in the hepatocyte of hepatic lobule first region at day1 after gene delivery,and there appeared a massive acidophilia degeneration of hepatocyte and apoptosis body,but no obvious inflammatory cell infiltration in correspending location.Transient noticeable increase of ALT/AST indicates that FasL-overexpressing hepatocytes may become effector cells to induce adjacent hepatocyte apoptosis.This provided the significant attempt to study further.It is essential to develop more effective expression system of FasL and enroll greater number of samples for futher elucidating the role and mechanism of hepatocyte death mediated by FasL/Fas in liver damage.