Inhibition Of Mice Peritoneal Fibrosis By PAMAM Vector Mediated PCTGF-shRNA

Continuous ambulatory peritoneal dialysis (CAPD) is well known to be an important substitute therapy for the patients in end stage renal disease (ESRD). Studies have shown that ultrafiltration failure (UFF) caused by dialysis related peritoneal fibrosis have become the main reason for the interruption of peritoneal dialysis in CAPD patients. It is urgently needed to find a new therapeutic tool against peritoneal fibrosis.Peritoneal fibrosis is a complicated process controlled by various cytokines. The transforming growth factor-beta (TGF-β) is one of the most studied cytokines and plays a central role in the initiation and progression of peritoneal fibrosis. However, as TGF-βis a pleiotropic cytokine with diverse targets and biologic functions essential in maintaining homeostasis, complete blockade of TGF-βsignaling pathways might lead to unexpected consequences. Therefore, Connective Tissue Growth Factor (CTGF), a downstream factor of TGF-βand specifically mediating TGF-β-induced fibrosis, is considered to be a better target to prevent peritoneal fibrosis. Our previous studies have shown that short hairpin RNA (shRNA) of CTGF mediated by a retroviral vector could significantly inhibit TGF-β1-induced expression of CTGF, VEGF and extracellular matrix in human peritoneal mesothelial cells, suggesting a potential approach in the treatment of peritoneal fibrosis. However, little is known about the application of CTGF RNA interference on peritoneal fibrosis animal model. Moreover, retroviral vector has a disadvantage of random insertion into host genome leading to less safety profile. Therefore, we are aiming at a more efficient gene therapy strategy to inhibit the expression of CTGF in peritoneal mesothelial cells.Recently, PAMAM dendrimers have received much attention as a new class of gene carriers. PAMAM is a non-virus nanometer-sized carrier with the advantages of high transfection efficiency and long-term stable expression without causing immunogenicity and genotoxicity. It has become a highly promising application in DNA vaccine and gene therapy research. In the present study, plasmid of CTGF-shRNA was constructed, delivered by PAMAM and transfected into mouse peritoneal mesothelial cells in vitro as well as mouse model of peritoneal fibrosis in vivo. Three specific aims are addressed in this thesis. Firstly, pGenesil-1 plasmid expressing CTGF-shRNA was constructed. Secondly, the functions of pCTGF-shRNA interference on primary mouse peritoneal mesothelial cells are investigated using PAMAM. Finally, the effects of pCTGF-shRNA interference on mouse peritoneal fibrosis model are investigated using PAMAM. The objective, methods, results and conclusions are listed here.Objective: To construct pGenesil-1 plasmid containing the CTGF- shRNA (pCTGF-shRNA). Transfect mouse peritoneal mesothelial cells in vitro and mouse peritoneal fibrosis model in vivo through PAMAM. Investigate the effects of pCTGF-shRNA on the expression of CTGF, TGF-β1 and FN in mouse peritoneal mesothelial cells or mouse peritoneum.Methodes: 1. Based on shRNA design strategy, two target sequences (mCTGF1and mCTGF2) and one negative control sequence (HK) were designed. These three double stranded DNAs were ligated to pGenesil-1 separately and three plasmids named pCTGF1-shRNA,pCTGF2-shRNA and pHK-shRNA were formed which were confirmed by endonuclease digestion and DNA sequencing. The stability of PAMAM G9/DNA compounds was also tested.2. Primary MPMCs were isolated from mouse peritoneum through trypsinization. Transfection of pCTGF1-shRNA,pCTGF2-shRNA and pHK-shRNA to MPMCs (3rd generation, induced by 4.25% high glucose) was performed using PAMAM G9. The expression of CTGF,TGF-β1 and FN mRNA was determined by semi-quantification reverse ranscription PCR (RT-PCR). The protein level of CTGF,TGF-β1 and FN was determined by Western Blot.3. Kunming mice were randomly divided into transfection group and control group and were injected with PAMAM G9/pHK-shRNA or saline respectively (intraperitoneal injection). Collect parietal and visceral peritoneum at 24h,48h,96h and 7d, and the expressions of GFP in different groups were detected by both fluorescence microscope and Western Blot.4. C57BL/6 mouse were randomly divided into four groups: CTGF RNA interference-treated group, HK transfection group, peritoneal fibrosis group and normal control group. After corresponding treatments for 28 days, the parietal and visceral peritoneum was collected. The thickness of parietal peritoneum was measured by Masson' dye staining. The expression of CTGF, TGF-β1 and FN mRNA was determined by semi-quantification RT-PCR. The protein of CTGF,TGF-β1 and FN was determined by Western Blot and immunohistochemical method.Results: 1. The pGenesil-1 plasmid containing CTGF-shRNA construct was confirmed by endonuclease digestion and DNA sequencing. The structure of PAMAM G9/DNA compounds was stable and PAMAM can protect the bound DNA from restriction enzyme digestion.2. With the treatment of 4.25% glucose, the expression of CTGF,TGF-β1 and FN in MPMCs were significantly up-regulated. Introduction of pCTGF1-shRNA and pCTGF2-shRNA resulted in significant reduction of CTGF and FN expression (P＜0.05) with pCTGF2-shRNA more effective. But the introduction of these three plasmids did not lead to significant reduction in TGF-β1 expression in MPMCs. 3. The GFP expression was not detected in normal control group. However, GFP expression was detected in PAMAM Gg/pHK-shRNA transfection group at different time point (24h,48h,96h,7d). Both Western Blot and tissue section showed that GFP expression peaks were at 48h.4. The average peritoneum thickness was (24.7±3.8)μm,(34.6±4.2)μm,(36.2±3.6)μm and(7.5±2.4)μm, in CTGF RNA interference-treated group, HK transfection group, peritoneal fibrosis group and control group, respectively. CTGF RNA interference-treated group had significantly thinner peritoneum compared to HK control group and peritoneal fibrosis group (P＜0.01) with still thicker peritoneum compared to the normal control group (P＜0.01). The peritoneal mRNA and protein expression levels of CTGF ,TGF-β1 and FN in CTGF RNA interference-treated group, HK control group and peritoneal fibrosis group were significantly increased compared to the normal control group (P＜0.05) with no difference between peritoneal fibrosis group and HK control group (P＞0.05). However, mRNA and protein levels of CTGF, TGF-β1 and FN in CTGF RNA interference-treated group were markedly decreased compared to peritoneal fibrosis group(P＜0.05).Conclusions: PAMAM has the ability to efficiently transfect pCTGF1-shRNA,pCTGF2-shRNA into primary mouse peritoneal mesothelial cells in vitro and has prominent inhibition effects on increased expression of CTGF and FN level induced by high glucose with higher inhibition by pCTGF2-shRNA. However, neither pCTGF1-shRNA nor pCTGF2- shRNA has any influence on the expression of TGF-β1 under the same circumstances.PAMAM can also efficiently transfect pCTGF2-shRNA into mouse peritoneal fibrosis model and result in inhibition on increased expression of CTGF, TGF-β1 and FN level in mouse peritoneum induced by high glucose dialysis fluid and inhibition in peritoneum thickening or collagen deposition. All these indicate that PAMAM vector mediated pCTGF- shRNA can be used as a very efficient strategy in gene therapy of peritoneal fibrosis.