| Primary liver cancer (PLC) is a common malignant tumor, although radical surgical resection and liver transplantation are still the main way to cure,80%of patients missed opportunity to radical surgery after diagnosis. More and more conclusive clinical data supported that the PLC is a radiosensitive tumor. Radiaiton induced liver disease(RILD) is mainly fatal complications secondary to radiotherapy. The liver is a radiation sensitive organ in vivo similar to bone marrow, lymphoid tissues, however, hepatocytes are radiation resistance in vitro. It is difficult to explain using the "classical targeted cells theory". Previous studies have confirmed that intraheptic microenvironment, including non-parenchymal cells and released cytokines play the pivotal role in pathogenesis of RILD, which is called radiation induced non-targeted effect. Radiation-induced multiple intracellular signaling pathways activation contribute to information transfer between cells form a large regulatory networks, but the relationship between them and the control model is still unclear. Literatures support nonparenchymal cells are radiation sensitive cells and released large amounts of cytokines after radiotherapy. It has been confirmed that transforming growth factor-p (TGF-β) is closely related with the RILD, but the exact mechanism remains unclear. This study concentrated on radiation-induced non-targeted effects through inhibiting TGF-βsignaling or Kupffer cells in the pathogenesis of RILD.Try to explore the molecular mechanism and prevention strategies of RILD from a new perspective.Part I:Construction of recombinant adenovirus carrying the human soluble TβRII-Fc fusion gene using the AdMax systemPurpose: To construct the adenovirus vector encoding the sequence of the type II TGF-βreceptor (TβRII) fused to the human immunoglobulin Fc fragment (IgFC).Methods:Human TβRII fusion gene were amplified by reverse transcriptase -polymerase chain reaction (RT-PCR). TβRII gene was cloned into pDC316-cmv-EGFP vector. Primers designing and PCR amplification for IgFC. Ligation for the PCR product and PDC316-TPRII to obtain PDC316-TβRII-IgFC. The positive plasmids are confirmed by complete sequencing. All gene sequences were confirmed by PCR verification, restriction digestion and sequencing.The PDC316-TβRII-IgFC plasmid was cotransfected with adenoviral backbone vectors pBHGlox_E1,3Cre into 293 cells to package therecombinant adenovirus. Extracted viral genomic DNA followed PCR verification the target gene. Product and purify virion, then schizolysis the purified viruses with 10×virus lysate buffer.Results:Recombinant adenoviral vector Ad TβRIIFC was constructed successfully, which was confirmed by restriction ellzyme digestion and GFP expression. Conclusion:The recombinant adenoviral vector carrying human TβRII-FC fusion gene was successfully constructed, and will be lay the foundation of application of gene therapy to radiation induced liver fibrosis.Part II:Radiation-Induced Liver Fibrosis is mitigated by gene therapy inhibiting transforming Growth Factor-βSignaling in the RatPurpose:We determined whether anti-TGF-βintervention can halt the progression of established radiation-induced liver fibrosis.Methods:A replication-defective adenoviral vector expressing the extracellular portion of human TβRII and the Fc portion of immunoglobulin IgG fusion protein (AdTβRIIFc) was produced. The entire liver was exposed to 30 Gy irradiation to generate a RILF model (RILFM). Then, RILFM animals were treated with AdTβRIIFc (1×1011 PFU, TβRII), control virus (1×1011 PFU, AdGFP), or saline (SALIN). Delayed radiation liver injury was assessed by histology and immunohistochemistry. Chronic oxidative stress damage, hepatic stellate cell (HSC) activation, and hepatocyte regeneration were also analyzed.Results:In rats infected with AdTβRIIFc, fibrosis was significantly mitigated compared with rats treated with AdGFP or saline, as assessed by histology, hydroxyproline content, and the serum level of hyaluronic acid. Compared with AdGFP rats, AdTβRIIFc-treated rats exhibited decreased oxidative stress damage and HSC activation, and preserved liver function.Conclusions:Our results demonstrate that TGF-βplays a critical role in the progression of liver fibrosis and suggest that anti-TGF-βintervention is feasible and ameliorates established fibrotic livers. In addition, chronic oxidative stress may be involved in the progression of RILF.Part III:Inactivation of kupffer cells by Gadolinium Chloride protects murine liver from radiation-induced apoptosis.Purpose:To determine whether the inhibition of Kupffer cells before radiotherapy (RT) would protect hepatocytesfrom radiation-induced apoptosis.Materials and Methods:A single 30-Gy fraction was administered to the upper abdomen of Sprague-Dawley rats.The Kupffer cell inhibitor gadolinium chloride (GdC13; 10 mg/kg body weight) was intravenously injected 24 h before RT. The rats were divided into four groups:group 1, sham RT plus saline (control group); group 2, sham RT plus GdC13; group 3, RT plus saline; and group 4, RT plus GdC13. Liver tissue was collected for measurementof apoptoic cytokine expression and evaluation of radiation-induced liver toxicity by analysis of liver enzyme activities, hepatocyte micronucleus formation, apoptosis, and histologic staining.Results:The expression of interleukin-lb, interleukin-6, and tumor necrosis factor-a was significantly attenuated in group 4 compared with group 3 at 2,6,24, and 48 h after injection (p<0.05). At early points after RT, the rats in group 4 exhibited significantly lower levels of liver enzyme activity, apoptotic response, and hepatocyte micronucleus formation compared with those in group 3.Conclusion:Selective inactivation of Kupffer cells with GdC13 reduced radiation-induced cytokine production andprotected the liver against acute radiation-induced damage.
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