Transforming growth factor-β1 short hairpin RNA inhibits renal allograft fibrosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Transforming growth factor-β1 (TGF-β1) is known to be a key fibrogenic cytokine in a number of chronic fibrotic diseases, including chronic allograft nephropathy. We examined the effects of inhibition of TGF-β1 expression by RNA interference on renal allograft fibrosis, and explored the mechanisms responsible for these effects.</p><p><b>METHODS</b>A Sprague-Dawley-to-Wistar rat model of accelerated kidney transplant fibrosis was used. Sixty recipient adult Wistar rats were randomly divided into four groups: group J (sham-operated group), group T (plasmid-transfected group), group H (control plasmid group), and group Y (transplant only group). Rats in group T were transfected with 200 µg of TGF-β1 short hairpin RNA (shRNA). Reverse transcription-polymerase chain reaction and Western blotting were used to examine the expression of TGF-β1, Smad3/7, E-cadherin, and type I collagen. The distribution of type I collagen was measured by immunohistochemistry. The pathologic changes and extent of fibrosis were assessed by hematoxylin and eosin and Masson staining. E-cadherin and α-smooth muscle actin immunohistochemical staining were used to label tubular epithelial cells and fibroblasts, respectively.</p><p><b>RESULTS</b>Plasmid transfection significantly inhibited the expression of TGF-β1, as well as that of its target gene, type I collagen (P < 0.05 and P < 0.01, respectively). In addition, the degree of fibrosis was mild, and its development was delayed in plasmid-transfected rats. In contrast, TGF-β1-shRNA transfection maintained the expression of E-cadherin in tubular epithelial cells while it inhibited the transformation from epithelial cells to fibroblasts. Blood urea nitrogen and serum creatinine were lower in the plasmid group than in the control groups (P < 0.05 and P < 0.01, respectively).</p><p><b>CONCLUSIONS</b>This study suggests that transfection of a TGF-β1-shRNA plasmid could inhibit the fibrosis of renal allografts. The mechanism may be associated with the downregulation of Smad3 and upregulation of Smad7, resulting in suppressed epithelial-myofibroblast transdifferentiation and extracellular matrix synthesis.</p>

Glycosyl-phosphatidylinositol-anchored interleukin-2 expressed on tumor-derived exosomes induces anti-tumor immune response / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2.</p><p><b>METHODS</b>To construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2.</p><p><b>RESULTS</b>The pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70, intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P<0.05).</p><p><b>CONCLUSIONS</b>GPI-IL-2 gene-modified tumor cells can make the exosomes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder transitional cell tumors.</p>

Preparation of renal cancer vaccine of IL-12-anchored exosomes and its antitumor effect in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.</p><p><b>METHODS</b>A mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.</p><p><b>RESULTS</b>Mammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.</p><p><b>CONCLUSION</b>A vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.</p>

Exosomes derived form bladder transitional cell carcinoma cells induce CTL cytotoxicity in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To isolate and purify exosomes derived from human bladder transitional cell carcinoma T24 cells, analyze the morphology and protein composition, and investigate the antitumor effect of specific cytotoxic T lymphocytes induced by exosomes.</p><p><b>METHODS</b>Exosomes were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and characterized by electron microscopy and Western blot. Dendritic cells were amplified and purified from peripheral blood and pulsed with exosomes. Then they were co-cultured with T cells, and divided into 3 groups: exosome-pulsed DC group, unplused DC group and control group. Alamar-Blue assay was used to evaluate the specific cytolytic activity.</p><p><b>RESULTS</b>The exosomes were in size about 30 approximately 90 nm saucer-shaped membranous vesicles. HSP70, ICAM-1 and CK20 were detected by Western blot. The CTL induced by DC pulsed with exosomes had significant cytolytic activity (P < 0.01).</p><p><b>CONCLUSION</b>The exosomes derived from T24 cells are loaded with immunoprotein HSP70 and ICAM-1, and DC pulsed with exosomes can promote the anti-tumor effect of CTLs in vitro.</p>

Proliferation apoptotic influence of crocin on human bladder cancer T24 cell line / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the proliferation, apoptosis and mechanisms on T24 cell of transitional cell carcinoma of bladder (TCCB) by crocin.</p><p><b>METHOD</b>MTT assay was used to evaluate the proliferation of T24 cells. The changes of cell cycle and cell apoptotic percentage were measured by flow cytometry. T24 cells were inoculated into BALB/c nude mice to establish model of carcinoma of bladder. The mice were randomly divided into control group and experimental group. After treatment with 50 mmol x L(-1) crocin, the inhibited growth of tumor was observed. Electronic microscope was used to observe the morphological changes. The expressions of Bcl-2, Bax, Survivin and Cyclin D1 were detected by immunohistochemistry.</p><p><b>RESULT</b>The growth of T24 cells was remarkably inhibited after treatment of crocin. Flow cytometric profiles revealed that crocin led to the increase of the cells in G0/G1 phase, the percentage of cell apoptosis was also increased. Crocin could inhibit the growth of BALB/c xenograft tumor. The morphology changes of cell apoptosis were observed. Bcl-2, Cyclin D1 and survivin expressions determined by immunohistochemical staining were down-regulated after treatment with Bax expression up-regulated.</p><p><b>CONCLUSION</b>Crocin exerts both in vitro and in vivo anti-cancer effect on TCCB T24 cell line. The mechanisms may change tumour cell cycle and induce tumour cell apoptosis by down-regulating the expression of Bcl-2, Survivin, Cyclin D1 and up-regulating the expression of Bax.</p>

Construction of deltaNp63 specific small hairpin RNA expressing plasmid and its role in bladder cancer--a preliminary study / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct deltaNp63 specific small hairpin RNA (shRNA) expressing plasmid,to examine its inhibitory effect to the expression of deltaNp63 protein and mRNA in transitional cell carcinoma of the bladder (TCCB) , its effect on TCCB cells cycle and proliferation.</p><p><b>METHODS</b>DeltaNp63 specific oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form double strand DNA fragments and this fragment was cloned into Pgenesil-1 plasmid. The recombinant deltaNp63-shRNA expression construct was confirmed by using Pst I + Sal I double digestion and by sequencing. Fluorescence staining was used to confirm the success of transfection in TCCB cells under the fluorescence microscope. The inhibitory effect of deltaNp63-shRNA construct was examined with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining assay. The cell cycle of TCCB cells was assayed by flow cytometry (FCM). The cellular proliferation of TCCB cells was assayed by tetrazolium bromide (MTT) colorimetry.</p><p><b>RESULTS</b>The deltaNp63-shRNA expression plasmid was successfully constructed and transfected into TCCB cells. It can effectively reduce the expression of deltaNp63 protein and mRNA. The reduction rate of deltaNp63 mRNA was 63.0%, and the G0/G1 ratio was increased and S phase was decreased in transfected TCCB cells. The cellular proliferation was also lower in transfected 5637 cells in comparrison with that of non-transfected TCCB cells.</p><p><b>CONCLUSION</b>A deltaNp63-shRNA expression plasmid, constructed from Pgenesil-1 plasmid, can successfully be transfected into TCCB cells and can effectively inhibit the expression of deltaNp63 protein and mRNA. It also can take part in regulation of the cell cycling and inhibit the cellular proliferation of TCCB cells.</p>

Effect of saffor (Carthamus tinctorius) injection on renal ischemia/reperfusion injury in rats / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect and mechanism of saffor injection on renal ischemia/reperfusion (I/R) injury in rats.</p><p><b>METHOD</b>Seventy-five SD rats were randomly divided into five groups (n = 15, in each), normal control groups, I/R control groups, low-dose treatment groups, middle-dose treatment groups and high-dose treatment groups. After rat's I/R injury model was established, renal function was assessed by measuring serum creatinine, blood urea nitrogen, urine osmotic pressure and urine osmotic pressure/blood osmotic pressure, the apoptosis rate in I/R renal tissure was measured by TUNEL method and caspase-3 concentration was measured by immunohistochemistry.</p><p><b>RESULT</b>Reperfusion of the ischemic kidney induced marked renal dysfunction. Saffor injection significantly inhibited the reperfusion-associated increase in apoptosis rate and caspase-3 protein absorbance value. Moreover, the renal dysfunction at all treatment groups was markedly ameliorated by Saffor injection. (P < 0.01).</p><p><b>CONCLUSION</b>The results show that saffor injection significantly reduces the renal dysfunction and injury caused by I/R of the kidney, And the protective effect of Saffor injection may be related to the inhibition of cell apoptosis and caspase-3 gene expression following renal I/R.</p>