Integrated proteogenomic characterization reveals an imbalanced hepatocellular carcinoma microenvironment after incomplete radiofrequency ablation.

BACKGROUND: Efforts to precisely assess tumor-specific T-cell immune responses still face major challenges, and the potential molecular mechanisms mediating hepatocellular carcinoma (HCC) microenvironment imbalance after incomplete radiofrequency ablation (iRFA) are unclear. This study aimed to provide further insight into the integrated transcriptomic and proteogenomic landscape and identify a new target involved in HCC progression following iRFA. METHODS: Peripheral blood and matched tissue samples were collected from 10 RFA-treated HCC patients. Multiplex immunostaining and flow cytometry were used to assess local and systemic immune responses. Differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were explored via transcriptomic and proteogenomic analyses. Proteinase-3 (PRTN3) was identified in these analyses. And then, the ability of PRTN3 to predict overall survival (OS) was assessed in 70 HCC patients with early recurrence after RFA. In vitro CCK-8, wound healing and transwell assays were conducted to observe interactions between Kupffer cells (KCs) and HCC cells induced by PRTN3. The protein levels of multiple oncogenic factors and signaling pathway components were detected by western blotting. A xenograft mouse model was built to observe the tumorigenic effect of PRTN3 overexpression on HCC. RESULTS: Multiplex immunostaining revealed no immediate significant change in local immune cell counts in periablational tumor tissues after 30 min of iRFA. Flow cytometry showed significantly increased levels of CD4+ T cells, CD4+CD8+ T cells, and CD4+CD25+CD127- Tregs and significantly decreased the levels of CD16+CD56+ natural killer cells on day 5 after cRFA (p < 0.05). Transcriptomics and proteomics revealed 389 DEGs and 20 DEPs. Pathway analysis showed that the DEP-DEGs were mainly enriched in the immunoinflammatory response, cancer progression and metabolic processes. Among the DEP-DEGs, PRTN3 was persistently upregulated and closely associated with the OS of patients with early recurrent HCC following RFA. PRTN3 expressed in KCs may affect the migration and invasion of heat stress-treated HCC cells. PRTN3 promotes tumor growth via multiple oncogenic factors and the PI3K/AKT and P38/ERK signaling pathways. CONCLUSIONS: This study provides a comprehensive overview of the immune response and transcriptomic and proteogenomic landscapes of the HCC milieu induced by iRFA, revealing that PRTN3 promotes HCC progression after iRFA. TRIAL REGISTRATION: ChiCTR2200055606, http://www.chictr.org.cn/showproj.aspx?proj=32588 .

miR-449a ameliorates acute rejection after liver transplantation via targeting procollagen-lysine1,2-oxoglutarate5-dioxygenase 1 in macrophages.

Acute rejection (AR) is an important factor that leads to poor prognosis after liver transplantation (LT). Macrophage M1-polarization is an important mechanism in AR development. MicroRNAs play vital roles in disease regulation; however, their effects on macrophages and AR remain unclear. In this study, rat models of AR were established following LT, and macrophages and peripheral blood mononuclear cells were isolated from rats and humans, respectively. We found miR-449a expression to be significantly reduced in macrophages and peripheral blood mononuclear cells. Overexpression of miR-449a not only inhibited the M1-polarization of macrophages in vitro but also improved the AR of transplant in vivo. The mechanism involved inhibiting the noncanonical nuclear factor-kappaB (NF-κB) pathway. We identified procollagen-lysine1,2-oxoglutarate5-dioxygenase 1 (PLOD1) as a target gene of miR-449a, which could reverse miR-449a's inhibition of macrophage M1-polarization, amelioration of AR, and inhibition of the NF-κB pathway. Overall, miR-449a inhibited the NF-κB pathway in macrophages through PLOD1 and also inhibited the M1-polarization of macrophages, thus attenuating AR after LT. In conclusion, miR-449a and PLOD1 may be new targets for the prevention and mitigation of AR.

XPA serves as an autophagy and apoptosis inducer by suppressing hepatocellular carcinoma in a PI3K/Akt/mTOR dependent manner.

BACKGROUND: To explore the potential biological function of XPA (Xeroderma pigmentosum group A) in hepatic neoplasms and the underlying molecular mechanisms. METHODS: Liver cells were used as experimental models to establish HCC (hepatocellular carcinoma) in vitro. Protein extractions were subjected to Western blotting to detect the proteins expression. The lentivirus transfection efficiency was confirmed by Western blot and RT-qPCR, Tunnel staining was used to detect apoptosis, and Transwell assays were used to observe cell migration and invasion. Cell proliferation was detected with colony formation and CCK-8 (cell counting kit-8) assays. RESULTS: XPA expression was obviously lower in HCC tissue and liver cancer cell lines. XPA overexpression induced autophagy and apoptosis by increasing LC3B II/I, Beclin1, cleaved-caspase-3, and Bax expression and decreasing p62 and Bcl2 protein levels. XPA also suppressed HCC EMT (Epithelial-Mesenchymal Transition) by increasing E-cadherin and decreasing N-cadherin and vimentin protein expression. Cell proliferation, migration and invasion in vivo were significantly inhibited by the overexpression of XPA, and p-PI3K, p-Akt, and p-mTOR expression were decreased in LV-XPA cells. In general, XPA inhibited HCC by inducing autophagy and apoptosis and by modulating the expression of PI3K/Akt/mTOR proteins. CONCLUSIONS: XPA overexpression was found to suppress HCC by inducing autophagy and apoptosis and repressing EMT and proliferation. Each of these effects may be involved in modulating the PI3K/Akt/mTOR signaling pathway.

Fisetin mitigates hepatic ischemia-reperfusion injury by regulating GSK3ß/AMPK/NLRP3 inflammasome pathway.

BACKGROUND: Hepatic ischemia-reperfusion (I/R) injury (IRI) represents a crucial challenge in liver transplantation. Fisetin has anti-inflammatory, anti-aging and anti-oxidative properties. This study aimed to examine whether fisetin mitigates hepatic IRI and examine its underlying mechanisms. METHODS: Sham or warm hepatic I/R operated mice were pretreated with fisetin (5, 10 or 20 mg/kg). Hepatic histological assessments, TUNEL assays and serum aminotransferase measurements were performed. An in vitro hypoxia/reoxygenation (H/R) model using RAW264.7 macrophages pretreated with fisetin (2.5, 5 or 10 µmol/L) was also used. Serum and cell supernatant concentrations of interleukin-1ß (IL-1ß), IL-18 and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Protein levels of p-GSK3ß, p-AMPK and NLR family pyrin domain-containing 3 (NLRP3)-associated proteins were detected by Western blotting. RESULTS: Compared with the I/R group, fisetin pretreatment reduced pathological liver damage, serum aminotransferase levels, serum concentrations of IL-1ß, IL-18 and TNF-α in the murine IRI model. Fisetin also reduced the expression of NLRP3 inflammasome-associated proteins (NLRP3, cleaved caspase-1, IL-1ß and IL-18) in I/R-operated liver. The experiments in vitro showed that fisetin decreased the release of IL-1ß, IL-18 and TNF-α, and reduced the expression of NLRP3 inflammasome-associated proteins in H/R-treated RAW264.7 cells. Moreover, fisetin increased the expressions of p-GSK3ß and p-AMPK in both models, indicating that its anti-inflammatory effects were dependent on GSK3ß/AMPK signaling. The anti-inflammatory effects of fisetin were partially inhibited by the AMPK specific inhibitor compound C. CONCLUSIONS: Fisetin showed protective effects against hepatic IRI, countering inflammatory responses through mediating the GSK3ß/AMPK/NLRP3 inflammasome pathway.

Integrating transcriptomes and somatic mutations to identify RNA methylation regulators as a prognostic marker in hepatocellular carcinoma.

BACKGROUND: RNA methylation modifying plays an important role in the occurrence and progression of a range of human cancers including hepatocellular carcinoma (HCC), which is characterized by a mass of genetic and epigenetic alterations. However, the treatment targeting these alterations is limited. METHODS: We used comprehensive bioinformatics analysis to analyze the correlation between cancer-associated RNA methylation regulators and HCC malignant features in network datasets. RESULTS: We identified two HCC subgroups (cluster 1 and 2), which had clearly distinct clinicopathological, biofunctional and prognostic characteristics, by consensus clustering. The cluster 2 subgroup correlated with malignancy of the primary tumor, higher tumor stage, higher histopathological grade and higher frequency of TP53 mutation, as well as with shorter survival when compared with cluster 1. Gene enrichment indicated that the cluster 2 correlated to the tumor malignancy signaling and biological processes. Based on these findings, an 11-gene risk signature was built, which not only was an independent prognostic marker but also had an excellent power to predict the tumor features. CONCLUSIONS: Our study indicated that RNA methylation regulators are vital for HCC malignant progression and provide an important evidence for RNA methylation, methylation regulators are actionable targets for anticancer drug discovery.

IL-27 is elevated in sepsis with acute hepatic injury and promotes hepatic damage and inflammation in the CLP model.

BACKGROUND: Immuno-inflammation plays an important role in the pathophysiological process of sepsis-associated acute hepatic injury (AHI). Interleukin 27 (IL-27) is an important inflammatory regulator; however, its role in this condition is not clear. METHODS: The clinical data and IL-27 serum levels in sepsis patients with or without AHI were analysed. Classical caecal ligation puncture (CLP) models were established in wild-type (WT) and IL-27 receptor (WSX-1)-deficient (IL-27R-/-) mice. In addition, exogenous IL-27 was injected into these mice, and the levels of IL-27, IL-6, and tumour necrosis factor alpha (TNF-α) in the serum and liver were then measured by enzyme-linked immunoassay (ELISA), quantitative PCR, and Western blotting. The severity of liver damage was evaluated by haematoxylin and eosin staining of liver tissue, TUNEL assay and evaluation of alanine aminotransferase (ALT) and aspartate transaminase (AST) serum levels. Furthermore, the effects of IL-27 on the levels of phosphorylated c-Jun N-terminal kinase (JNK) in macrophages were assessed by Western blotting, and the effects of IL-27 on the expression of IL-6 and TNF-α in macrophages were assessed by ELISA. RESULTS: IL-27 was elevated in sepsis patients with acute hepatic injury, which correlated with the Acute Physiologic Assessment and Chronic Health Evaluation II (APACHEII) scores, Sequential Organ Failure Assessment (SOFA) scores, and procalcitonin, C-reactive protein, IL-6, and TNF-α expression. In the CLP-WT group, IL-27 was highly expressed in the serum and liver, which correlated with the elevated content of ALT, AST, TNF-α, IL-6, and p-JNK in the serum and liver and the pathological injury of the liver. In CLP-IL-27R-/- group, however, the levels of ALT, AST, TNF-α, IL-6, and p-JNK in the serum and liver and the pathological injury of the liver were decreased. Treatment with exogenous IL-27 led to a further increase in these cytokines in WT mice after CLP. IL-27 treatment and lipopolysaccharide stimulation in vitro increased the expression of p-JNK, IL-6, and TNF-α in macrophages, and these changes were decreased by a JNK signalling pathway inhibitor. CONCLUSION: IL-27 is elevated in sepsis patients, especially those with acute hepatic injury. In addition, IL-27 can promote inflammatory reactions in the CLP-induced hepatic injury mice model.

ß-catenin deficiency in hepatocytes aggravates hepatocarcinogenesis driven by oncogenic ß-catenin and MET.

Both activating and inactivating mutations in catenin ß1 (ctnnb1), which encodes ß-catenin, have been implicated in liver tumorigenesis in humans and mice, although the underlying mechanisms are not fully understood. Herein, we show that deletion of endogenous ß-catenin in hepatocytes aggravated hepatocellular carcinoma (HCC) development driven by an oncogenic version of ß-catenin (CAT) in combination with the hepatocyte growth factor receptor MET proto-oncogene receptor tyrosine kinase (MET). Although the mitogenic signaling and cell cycle progression was modestly impaired after CAT/MET transfection, the ß-catenin-deficient livers displayed changes in transcriptomes, increased DNA damage response, expanded Sox9+ cells, and up-regulation of protumorigenic cytokines, including interleukin-6 and transforming growth factor ß1. These events eventually exacerbated CAT/MET-driven hepatocarcinogenesis in ß-catenin-deficient livers, featured by up-regulation of extracellular signal-regulated kinase (Erk), protein kinase B (Akt), and Wnt/ß-catenin signaling and cyclin D1 expression. The resultant mouse tumors showed similar transcriptomes to human HCC samples with concomitant CTNNB1 mutations and MET overexpression. CONCLUSION: These data argue that while dominantly activating mutants of ß-catenin are oncogenic, inhibiting the oncogenic signaling pathway generates a pro-oncogenic microenvironment that may facilitate HCC recurrence following a targeted therapy of the primary tumor. An effective therapeutic strategy must require disruption of the oncogenic signaling in tumor cells and suppression of the secondary tumor-promoting stromal effects in the liver microenvironment. (Hepatology 2018;67:1807-1822).

IL-37 induces autophagy in hepatocellular carcinoma cells by inhibiting the PI3K/AKT/mTOR pathway.

Autophagy is an intracellular "self-eating" process that is closely related to inflammation and cellular immunity. New studies indicate that autophagy is also involved in tumor suppression. The anti-inflammatory cytokine interleukin-37 (IL-37) has been shown to have tumor-suppressive abilities in hepatocellular carcinoma (HCC). Notably, autophagy appears to play a dual role in the development of HCC and may be involved in both tumorigenesis and tumor suppression. However, the potential role of IL-37 in autophagy is currently unknown. In this study, we investigated the effect of IL-37 on autophagy in multiple HCC cell lines. In doing so, we found that IL-37 inhibits proliferation in HCC cells and also induces autophagy and apoptosis in the SMMC-7721 and Huh-7 cell lines. Further experiments revealed that IL-37 treatment reduced the levels of phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated p70 ribosomal protein s6 kinase (p-p70S6K) and phosphorylated 4E-binding protein 1 (4E-BP1). Moreover, treatment with an AKT agonist, insulin-like growth factor 1 (IGF-1), reversed these IL-37-mediated effects on autophagy, and treatment with an phosphoinositide-3-kinase (PI3K)/AKT inhibitor, LY294002, mimicked the effects of IL-37. Taken together, these results indicate that IL-37 regulates autophagy in SMMC-7721 and Huh-7 cells via inhibition of the PI3K/AKT/mTOR signaling pathway.

Resveratrol preconditioning protects hepatocytes against hepatic ischemia reperfusion injury via Toll-like receptor 4/nuclear factor-κB signaling pathway in vitro and in vivo.

The purpose of this study was to investigate the protective effect of resveratrol against hepatic ischemia reperfusion injury (HIRI) and explore the potential underlying mechanism. Resveratrol-pretreated BRL-3A (rat liver) cells and rats underwent hypoxia/reoxygenation and hepatic ischemia/reperfusion, respectively. BRL-3A cell damage was evaluated, and the mRNA and protein expression of related signal molecules was assessed in cell model. The protein expression of related signal molecules was also assessed in rat model. Inflammatory cytokines levels were determined in the cell supernatant and rat serum while rat liver function and hepatocyte apoptosis were assessed. The results revealed that resveratrol significantly enhanced cell viability, inhibited cell apoptosis, and decreased levels of lactate dehydrogenase (LDH) and production of tumor necrosis factor-α (TNF-α) and interleukin-(IL)-1ß in the cell supernatant. In addition, resveratrol ameliorated elevated Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB, and the depressed inhibitor of NF-κB (IκB)-α caused by hypoxia/reoxygenation stimulation in BRL-3A cells. Moreover, resveratrol inhibited the translocation of NF-κB p65 after the stimulation of hypoxia/reoxygenation in BRL-3A cells. In vivo assays revealed that resveratrol reduced levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and liver pathological changes, while it alleviated hepatocyte apoptosis, negatively mediated the production of TNF-α and IL-1ß in serum, and reversed TLR4/NF-κB signaling pathway caused by hepatic ischemia/reperfusion stimulation in liver tissues. The results indicate that resveratrol protected hepatocytes against HIRI, which may be mediated in part via the TLR4/NF-κB signaling pathway.

Everolimus in de novo liver transplant recipients: a systematic review.

BACKGROUND: Everolimus has no nephrotoxicity and is used to treat patients with post-liver transplant chronic renal insufficiency. The present systematic review was to evaluate the efficacy and safety of everolimus in de novo liver transplant patients. DATA SOURCES: Randomized controlled trials comparing everolimus for de novo liver transplant in PubMed, the Cochrane Library, and ScienceDirect published up to March 31, 2014 were searched by two independent reviewers. Mean differences and 95% confidence interval (95% CI) for renal function, relative risk (RR) and 95% CI for treated biopsy-proven acute rejection (tBPAR), graft loss, death, neoplasms/tumor recurrence, and adverse events were collected. Meta-analyses were performed with RevMan version 5.10. RESULTS: A total of four randomized controlled trials covering 1119 cases were included. The meta-analyses revealed that compared with standard exposure of calcineurin inhibitors (CNIs), everolimus combined with reduced CNIs improved creatinine clearance (calculated with the Cockcroft-Gault formula) by 5.13 mL/min at one year (95% CI: 0.42-9.84; P=0.03), and decreased tBPAR (RR: 0.56; 95% CI: 0.35-0.90; P=0.02). Everolimus initiation with CNIs elimination improved glomerular filtration rate (GFR, measured with the modification of diet in renal disease formula) of 10.42 mL/min/1.73 m2 (95% CI: 3.44-17.41; P<0.01) one year after treatment, but increased tBPAR (RR: 1.71; 95% CI: 1.15-2.53; P<0.01). Everolimus decreased the risk of neoplasms/tumor recurrence after liver transplant (RR: 0.60; 95% CI: 0.34-1.03; P=0.06), but was associated with greater risk of adverse events which resulted in drug discontinuation (RR: 1.98; 95% CI: 1.49-2.64; P<0.01). CONCLUSIONS: Early introduction of everolimus combined with low-dose or no CNI in de novo liver transplant significantly improves renal function one year post treatment. Everolimus combined with low-dose CNI decreases the risk of tBPAR one year after liver transplant, but everolimus administered without CNIs increases tBPAR.

[Zinc finger protein designed to hepatitis B virus core promoter inhibit the transcription of HBV in HepG2.2.15 cells].

AIM: To observe the inhibition of hepatitis b virus(HBV) transcription in vitro by zinc fingrer protein(ZFP) which was designed specifically to bind DNA sequence in the HBV core promoter(Cp). METHODS: The sequence of HBV Cp was submitted to the Zinc Finger Tools and selected a sequence from the Cp as the target site of ZFP. The nucleic acid sequence of ZFP was synthesised and cloned into pEGFP-N1 or pcDNA3.1(+) to construct the recombinant plasmid pEGFP-N1/ZFP-Flag or pcDNA3.1(+)/ZFP. COS-7 cells were transfected with pEGFP-N1 or pEGFP-N1/ZFP-Flag, the expression of ZFP was observed by green fluorescent microscope, RT-PCR and Western blot. HepG2.2.15 cells were transfected with pcDNA3.1(+) or pcDNA3.1(+)/ZFP, HBeAg and HBV DNA levels in cell supernatant were detected by ELISA and FQ-PCR, HBV mRNA was tested by RT-PCR. RESULTS: ZFP can express in COS-7 cells.In th presence of ZFP, HBeAg expression and HBV DNA level was significantly reduce(P<0.05), and HBV mRNA was dramatically decreased. CONCLUSION: ZFP can express in eukaryotic cells and inhibit the transcription of HBV in vitro.

Constructing the HBV-human protein interaction network to understand the relationship between HBV and hepatocellular carcinoma.

BACKGROUND: Epidemiological studies have clearly validated the association between hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). Patients with chronic HBV infection are at increased risk of HCC, in particular those with active liver disease and cirrhosis. METHODS: We catalogued all published interactions between HBV and human proteins, identifying 250 descriptions of HBV and human protein interactions and 146 unique human proteins that interact with HBV proteins by text mining. RESULTS: Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HBV are made up of core proteins that are interconnected with many pathways. A global analysis based on functional annotation highlighted the enrichment of cellular pathways targeted by HBV. CONCLUSIONS: By connecting the cellular proteins targeted by HBV, we have constructed a central network of proteins associated with hepatocellular carcinoma, which might be to regard as the basis of a detailed map for tracking new cellular interactions, and guiding future investigations.

Modified arterialization of orthotopic liver transplantation in a mouse model.

BACKGROUND: With the establishment of genetically modified and gene knock-out models, the mouse has become an important animal model for liver transplantation. We examined hepatic rearterialization after liver transplantation in a mouse model. METHODS: Orthotopic liver transplantation was performed in 70 mice and sham-operation was performed in a control group of 40 mice. Based on the "two-cuff" method, a continuous suture approach was applied to the suprahepatic inferior vena cava and a cuff approach to the portal vein and the infrahepatic inferior vena cava. A biliary stent was inserted into the bile duct. The hepatic artery was reconstructed with end-to-side anastomosis. The survival rate of recipients was monitored at 24 hours, one week, and one month after the operation. Liver function and morphology were evaluated one month postoperatively. RESULTS: Postoperative survival rates were 94.3% at 24 hours, 91.4% at one week, and 85.7% at one month. No significant difference was seen between the experimental and control groups in liver function. The hepatic tissue preserved normal structure. CONCLUSION: Owing to its high survival rate and stability, this surgical approach is ideal for establishing an orthotopic liver transplantation mouse model with hepatic artery reconstruction.

[Effects of co-expression plasmid of human tPA and VEGF165 on the proliferation of vascular cells and fibrinolysis activity].

OBJECTIVE: To observe the expression of the co-expression plasmid of tissue-plasminogen activator (tPA) and vascular endothelia growth factor165 (VEGF165) in vascular smooth muscle cells (VSMC) and to study the effect of expressing products on the proliferation of VEC and VSMC and fibrinolysis activity. METHODS: The co-expression plasmid of tPA and VEGF165 (pBudCE4.1/tPA-VEGF165) was transfected into VSMC with the lipofection. The expression of tPA and VEGF165 at mRNA level was detected by RT-PCR and the protein level expression was detected by enzyme linked immunosorbent assay (ELISA). The fibrinolysis activity of culture medium of VSMC transfected tPA and VEGF165 genes was detected by fibrin plate technique. The VEC and VSMC were cultured with culture medium of VSMC transfected tPA and VEGF165 genes. And the proliferation of VEC and VSMC was evaluated with monoterazolium (MTT) and flow cytometry (FCM). RESULTS: The expression of tPA and VEGF165 at mRNA and protein levels in the transfected VSMC was demonstrated by RT-PCR and ELISA, respectively. The VSMC culture medium of transfected genes possessed evidently fibrinolysis activity. The expression products of tPA and VEGF165 in the VSMC had an evident effect on the VEC proliferation. But it had not an effect on the VSMC proliferation. CONCLUSION: The eukaryotic co-expression plasmid of tPA and VEGF165 can be expressed in transfected VSMCs. The expression products have an obvious biological activity. The present study lay the foundation for future making use of tPA and VEGF165 to prevent and treat vascular stenosis in transplanted heart.

[To study the effects of local co-transfection vascular endothelial growth factor165 and tissue-type plasminogen activator genes on inhibiting intimal hyperplasia after operation injury artery in rabbits].

OBJECTIVE: To observe the effects of local co-transfection vascular endothelial growth factor165 (VEGF165) and tissue-type plasminogen activator genes on inhibiting intimal hyperplasia and restenosis in rabbits artery after operation injury and possible mechanisms. METHODS: Micrology operation injury was used to establish the model of intimal injury of right external iliac artery in rabbits. To select 120 male New Zealand rabbits and were randomly divided into 3 groups (n = 40, in each group): Group A (physiological brine control group), Group B (pBudCE4.1 group), Group C (pBudCE4.1/VEGF165-tPA group). The vas-wall of micrology operation injury were infused respectively physiological brine, pBudCE4.1 and pBudCE4.1/VEGF165-tPA transfection solution by micro-injector. Each group were divided into 5 subgroups (n = 8, in each subgroup) randomly according to the sacrifice times (2 d, 1 week, 2 week, 4 week and 8 week after operation). The injured vascular specimen were harvested for pathology test, electric microscopy study, reverse transcription-PCR examining and immunochemistry detecting. RESULTS: The intimal area and narrow ratio of vases in Group C at every time point after operation were significantly lessened than that in Group A and Group B (P < 0.01). The narrow ratio of vases in Group C at 8 week after operation were decreased respectively by 57.9% and 59.0% than that in Group A and B. The expression of VEGF165 mRNA in Group C were increased significantly than that in Group A and B at every time point after operation (P < 0.01), the expression reached the peak at 1 week and continued to 4 week after operation. Immunohistochemical identified that tPA positive cell in Group C were significantly increased than that in Group A and B (P < 0.01) at every time point after operation. CONCLUSION: Local co-transfection VEGF165 and tPA genes could restrain intimal hyperplasia and restenosis of vas, which lay a foundation for future multi-gene therapy of vascular intimal hyperplasia.

Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells.

BACKGROUND: After organ transplantation, rapid repair of injured vascular endothelial cell(VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165(VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the proliferation of VEC. METHODS: The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme ( Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS: The RT-PCR product of the VEGF165 gene was about 576 bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS: pBudCE4.1/VEGF165 is successfully constructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.

[Construction of co-expression plasmid containing tPA and VEGF165 genes and its expression in VSMCs].

OBJECTIVE: To construct the eukaryotic co-expression plasmid of tissue-plasminogen activator (tPA) genes and vascular endothelia growth factor165 (VEGF165) and observe its expression in vascular smooth muscle cells (VSMCs). METHODS: The tPA and VEGF165 genes were cloned from hominal heart tissue with RT-PCR, and then the tPA and VEGF165 genes were cloned into eukaryotic expression plasmid pBudCE4.1 to construct the eukaryotic co-expression plasmid pBudCE4.1/tPA-VEGF165. The pBudCE4.1/tPA-VEGF165 was transfected into VSMCs via lipofection mediation. The expression levels of tPA and VEGF165 mRNAs in the transformed VSMCs were detected by RT-PCR and the expression levels of tPA and VEGF165 proteins were detected by ELISA. RESULTS: The size of RT-PCR product of tPA and VEGF165 genes was 1.9 kb and 576 bp respectively. Restrictive enzyme digestion analysis showed that recombinant co-expression plasmid pBudCE4.1/tPA-VEGF165 had been constructed successfully. The expression of tPA and VEGF165 at mRNA and protein levels in the transformed VSMCs and cells culture supernatant were detected respectively by RT-PCR and ELISA. CONCLUSION: The recombinant eukaryotic co-expression plasmid pBudCE4.1/tPA-VEGF165 has been successfully constructed. The tPA and VEGF165 are expressed in transformed VSMCs.

[The study on platelet-derived growth factor and proliferating cell nuclear antigen antisense oligodeoxynucleotides together inhibiting the stenosis of transplanted vascular].

OBJECTIVE: To study the effect of platelet-derived growth factor (PDGF) and proliferating cell nuclear antigen (PCNA) antisense oligodeoxynucleotides (AODN) together on inhibiting the proliferation of the stenosis of transplanted vascular. METHODS: The left and right external iliac arteries (length 1.0 cm) of rabbits were transplanted reciprocally. The transplanted vascular were respectively soaked in liposomes, PDGF-AODN, PCNA-AODN and PDGF-AODN adding PCNA-AODN solution about 20 minute, the vascular anastomotic were sutured by 8/0 suture of soaked in AODN solution. Four weeks later, the specimens were harvested for microscopy. The pathological morphology of transplanted vascular were observed under microscope (HE). The intimal thickness and area, stenosis ratio(%) of transplanted vascular were calculate and analysed statistically among group by computer system. The number of positive cells of PDGF's mRNA in transplanted vascular wall were counted with in situ hybridization histo-cytochemistry and the number of positive cells of PCNA's protein in transplanted vascular wall were counted by S-P immunochemistry. RESULTS: The intimal thickness and area, stenosis ratio of transplanted vascular, the number of PDGF and PCNA positive cell in PDGF-AODN adding PCNA-AODN group were significantly lower than those in other group (P < 0.01), and that were lower evidently than PDGF-AODN group and PCNA-AODN group. CONCLUSION: PDGF and PCNA antisense oligodeoxynucleotides together could significantly inhibit the proliferation of vascular smooth muscle cell and stenosis of transplanted vascular.

Protective effect of ecdysterone on PC12 cells cytotoxicity induced by beta-amyloid25-35.

OBJECTIVE: To examine the protective effect of ecdysterone (ECR) against beta-amyloid peptide fragment(25-35) (Abeta(25-35))-induced PC12 cells cytotoxicity, and to further explore its mechanism. METHODS: Experimental PC12 cells were divided into the Abeta group (treated by Abeta(25-35) 100 micromol/L), the blank group (untreated), the positive control group (treated by Vit E 100 micromol/L after induction) and the ECR treated groups (treated by ECR with different concentrations of 1, 50 and 100 micromol/L). The damaged and survival condition of PC12 cells in various groups was monitored by lactate dehydrogenase (LDH) release and MTT assay. The content of malondialdehyde (MDA) was measured by fluorometric assay to indicate the lipid peroxidation. And the antioxidant enzymes activities in PC12 cells, including superoxide dismutases (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), were detected respectively. RESULTS: After PC12 cells were treated with Abeta(25-35) (100 micromol/L) for 24 hrs, they revealed a great decrease in MTT absorbance and activity of antioxidant enzymes, including SOD, CAT and GSH-Px as well as a significant increase of LDH activity and MDA content in PC12 cells (P < 0.01). When the cells was pretreated with 1-100 micromol/L ECR for 24 hrs before Abeta(25-35) treatment, the above-mentioned cytotoxic effect of Abeta(25-35) could be significantly attenuated dose-dependently, for ECR 50 micromol/L, P < 0.05 and for ECR 100 micromol/L, P < 0.01. Moreover, ECR also showed significant inhibition on the Abeta(25-35) induced decrease of SOD and GSH-Px activity, but not on that of CAT. CONCLUSION: ECR could protect PC12 cells from cytotoxicity of Abeta(25-35), and the protective mechanism might be related to the increase of SOD and GSH-Px activities and the decrease of MDA resulting from the ECR-pretreatment.