Expression of Sonic hedgehog pathway-related genes in kidney tissues of rats with unilateral ureteral obstruction

AIM: To investigate the role of Sonic hedgehog (Shh) signaling pathway in renal interstitial fibrosis in the rats with unilateral ureteral obstruction (UUO). METHODS: Forty-eight male Sprague-Dawley rats were divided randomly into sham operation group and UUO model group with 24 rats each. The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fiber in the kidneys was detected with HE and Masson staining. Immunohistochemical analysis was performed to evaluate the expression of Shh signaling pathway-related proteins, including Shh, Smo,Ptch1 and Gli1. The contents of TGF-β₁ and Shh in the kidney tissues were determined by ELISA. Real-time RT-PCR was used to detect the mRNA expression of TGF-β₁, Col I, Col III and Shh signaling-related genes.RESULTS: Fibrosis observed with HE and Masson staining was obviously increased in UUO kidneys, and aggravated as time prolonged. The contents of TGF-β₁, Col I and Col III were also increased. In addition, the expression of Shh, Smo and Gli1 was markedly increased in obstructive kidneys, and the expression of Ptch1 was decreased (P<0.01), suggesting that Shh signaling was activated. The level of Shh in UUO rats was associated with the content of TGF-β₁. CONCLUSION: Shh signaling is activated in the progress of renal interstitial fibrosis in UUO rats, and the possible mechanism triggering the fibrogenic response is that Shh signaling promotes the expression of TGF-β₁.     

Effect of VDUP-1 on apoptosis of renal tubular epithelial cells induced by high glucose

AIM: To investigate the effect of vitamin D3 up-regulated protein 1 (VDUP-1) on apoptosis of renal tubular epithelial cells induced by high glucose and its mechanism. METHODS: Human renal proximal tubular epithelial cell line HK-2 was treated with high glucose. The mRNA and protein levels of VDUP-1 in HK-2 cells were detected by real-time PCR and Western blot. HK-2 cells were transfected with VDUP-1 small interfering RNA (siRNA). Real-time PCR and Western blot were used to detect the inhibitory effect. The HK-2 cells were treated with high glucose, and the change of VDUP-1 expression was detected. The apoptosis was analyzed by flow cytometry. The activities of caspase-3 and caspase-9 in the cells were measured. The tumor necrosis factor-α (TNF-α) content in the culture supernatant was examined by ELISA. The key proteins of Sonic hedgehog (Shh) signaling pathway, Patched 1 (Ptch1), Smoothened (Smo), zinc finger protein Gli2 and Shh, were determined by Western blot. The HK-2 cells were treated with exogenous Shh, and the levels of Ptch1, Smo and Gli2 were detected by Western blot. After the HK-2 cells with VDUP-1 silencing were treated with exogenous Shh and high glucose, the apoptosis was analyzed by flow cytometry, the activities of caspase-3 and caspase-9 in the cells were examined, and the TNF-α content in culture supernatant was measured by ELISA. RESULTS: High levels of VDUP-1 mRNA and protein were observed in the HK-2 cells treated with high glucose. The mRNA and protein levels of VDUP-1 were decreased in the HK-2 cells transfected with VDUP-1 siRNA(P<0.05). Compared with the normally cultured cells, the apoptotic rate of HK-2 cells was increased after high glucose treatment, and the activities of caspase-3 and caspase-9 and the content of TNF-α were also significantly increased (P<0.05). After down-regulation of VDUP-1 expression by siRNA transfection, the apoptotic rate of HK-2 cells decreased after high glucose treatment, and the activities of caspase-3 and caspase-9, and the content of TNF-α were also significantly decreased (P<0.05). The protein levels of Ptch1, Smo, Gli2 and Shh were decreased after high glucose culture, while down-regulation of VDUP-1 partly antagonized the effect of high glucose on the expression of Ptch1, Smo, Gli2 and Shh in the HK-2 cells. Exogenous Shh promoted the expression of Ptch1, Smo and Gli2, and inhibited the apoptosis of the HK-2 cells induced by high glucose. Exogenous Shh and down-regulation of VDUP-1 synergistically inhibited high glucose-induced apoptosis of the HK-2 cells. CONCLUSION: Down-regulation of VDUP-1 expression inhibits high glucose-induced apoptosis and release of TNF-α in renal tubular epithelial cells by activating Shh signaling pathway.     

Effect of cyclopamine on aristolochic acid-induced phenotypic transformation and Hedgehog pathway in renal epithelial cells

AIM: To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transformation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E. METHODS: NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at concentrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10 μmol/L). After cultured for 24 h, the mRNA expression of Ptch1, Smo, α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR. The protein levels of Shh and TGF-β1 were detected by ELISA. Immunofluorescence staining was used to evaluate the expression of Ptch1, Smo, α-SMA, E-cadherin and type III collagen in the NRK-52E cells. RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling, and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells. Treatment with cyclopamine inhibited HH signaling by decreasing Smo expression and increasing Ptch1 expression. Moreover, cyclopamine also down-regulated the expression of TGF-β1, α-SMA, type I collagen and III collagen, and up-regulated the expression of BMP-7, ZO-1 and E-cadherin. CONCLUSION: AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells, which can be inhibited by cyclopamine treatment. The possible mechanism is that cyclopamine suppresses the activation of HH signaling, resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.     

Effect of Sonic Hedgehog signaling blockade on growth of hepatocarcinoma cells

AIM: To investigate the effect of Sonic Hedgehog (Shh) signaling blockade on the growth of hematocarcinoma cells and underlying mechanisms. METHODS: The expression of Shh signaling molecules in hematocarcinoma cell lines BEL-7402, Huh7 and HepG2 was detected by RT-PCR. The cell viability was detected by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of apoptosis-related proteins was determined by Western blot. RESULTS: Shh signaling molecules were all expressed in BEL-7402, Huh7 and HepG2 cells. The mRNA expression of Patched (Ptch), Gli1 and Gli2 was down-regulated by anti-Shh antibody. Blockade of Shh signaling pathway inhibited the proliferation of hepatocarcinoma cells with increasing cells in G₀/G₁ phase and induced the apoptosis of hepatocarcinoma cells. Treatment with anti-Shh antibody down-regulated the protein expression of pro-caspase-3, pro-caspase-8 and pro-caspase-9, while up-regulated the protein levels of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 in BEL-7402 cells. CONCLUSION: Blockade of Shh signaling pathway inhibits the growth of hepatocarcinoma at different levels by cell cycle arrest and inducing apoptosis of hematocarcinoma cells.     

Expression of Smoothened and its role in endothelial cells in synovial tissues of rheumatoid arthritis

AIM: To investigate the expression of Sonic Hedgehog signaling pathway-associated factor Smoothened (Smo) and its role in endothelial cells in synovial tissue of active rheumatoid arthritis (RA). METHODS: Smo expression in synovial tissue from 4 RA patients and 4 patients with trauma or meniscal injury (without arthritis, used for control) was detected by the method of immunohistochemistry. Human umbilical vein endothelial cell line EA.hy926 was used as the model of synovial vascular endothelial cells. The expression of Smo was detected by Western blotting after TNF-α treatment. The small interfering RNA (siRNA) specifically targeting Smo gene was synthesized and transfected into EA.hy926 cells. The interference efficiency of the siRNA on the production of Smo protein was determined by Western blotting. The cells were treated with TNF-α and actinomycin D (ActD) 24 h after siRNA transfection. The cell survival rate was determined by CCK-8 assay and the apoptotic rate was examined by flow cytometry. RESULTS: Smo was highly expressed in synovial tissue from active RA patients, especially in endothelial cells as compared with control group. TNF-α significantly increased the protein expression of Smo in EA.hy926 cells. EA.hy926 cells transfected with Smo-siRNA showed a significant decrease in the cell viability with the cell survival rate of (24.30±0.45)% and the apoptotic rate of (48.00±1.96)%, as compared with those in negative control group ［(36.86±0.62）% and （31.70±0.82）%, respectively］. CONCLUSION: Smo may play a role in the regulation of apoptosis in endothelial cells in RA synovium.     

Effect of up-regulation of Shh-Gli1 signaling pathway on radioresistance and cell cycle in human esophageal cancer cell line Eca109

AIM:To investigate the relationship between Sonic Hedgehog (Shh) signaling pathway and cell cycle and radioresistance of esophageal cancer by up-regulating Gli1, a key factor in Shh signaling pathway. METHODS:The human esophageal cancer cell line Eca109 was transfected with plasmid to induce Gli1 over-expression, which served as Eca109-ox-Gli1 group. In addition, Eca109 cells transfected with empty plasmid served as negative control group and the untreated Eca109 cells were used as normal control group. The expression of Gli1 was confirmed by real-time PCR and Western blot. The radiosensitivity of the cells in the 3 groups was determined by colony formation assay. The effect of irradiation on the cell cycle was analyzed by flow cytometry. RESULTS:The expression of Gli1 in Eca109-ox-Gli1 group was higher than that in the other 2 groups (P<0.05). The survival fraction at dose of 2 Gy in Eca109-ox-Gli1 group was higher than that in normal control group, indicating that the radioresistance of the Eca109 cells transfected with Gli1 plasmid was increased. The cells in Eca109-ox-Gli1 group showed higher S phase proportion than that in normal control group and negative control group (P<0.01). After irradiation at dose of 6 Gy, all cells in the 3 groups found that the cell cycle was arrested at G₂/M phase, while the cells in normal control group showed higher G₂/M phase proportion than that in Eca109-ox-Gli1 group (P<0.01). CONCLUSION:The up-regulation of Gli1 may enhance the radioresistance of esophageal cancer by regulating the cell cycle.     

Prolactin induces interleukin-6 expression in PBMCs of rheumatoid arthritis patients

ATM: To investigate the correlation between serum prolactin (PRL) levels and disease activity in rheumatoid arthritis (RA) patients, and the regulatory role of PRL in interleukin-6 (IL-6) release from peripheral blood mononuclear cells (PBMCs), and to explore the MAPK-related mechanism of IL-6 release in PBMCs. METHODS: The clinicopathologic and hematologic parameters of 40 new-onset RA patients in the Department of Rheumatology of our hospital between March and September 2015 were collected. Chemilumineseent immunoassay (CLIA) was used to detect the serum PRL levels in the 40 RA patients and 20 healthy controls. The levels of IL-6 secretion by the PBMCs were evaluated using ELISA. Quantitative real-time PCR was employed to examine the mRNA expression of prolactin receptor (PRLR). MAPK pathway protein p-p38 levels were evaluated by Western blot. RESULTS: Serum PRL level in the RA patients was significantly higher than that in the healthy controls (P<0.01). Serum PRL level in active RA patients was significantly higher than that in inactive RA patients (P<0.01). Serum PRL level was positively correlated with DAS28, ESR and CRP (P<0.01). The expression of PRLR in the PBMCs was markedly increased in the RA patients than that in the healthy samples (P<0.01). Exposure of the PBMCs to PRL in the culture increased the release of IL-6, which was abolished by PRLR gene silencing or blocking the MAPK pathway.CONCLUSION: Serum PRL level is related to DAS28, ESR and CRP of RA patients and could be used as a predictor of disease activity. PRL/PRLR-p38 MAPK-IL-6 pathway may play a central role in the pathogenesis of RA.     

Expression and significance of hypoxia-inducible factor-1α on synovium from collagen-induced arthritis

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α) in the pathogenesis of rheumatoid arthritis (RA).METHODS: The collagen-induced arthritis (CIA) model was set up in male Wistar rats.The arthritis activity indexes,pathological changes and expression of HIF-1α on synovium at different time were observed through HE and immunohistochemistry staining.The correlations of HIF-1α expression with arthritis activity indexes and pathological scores were analyzed.RESULTS: Cytoplasmic and nucleic HIF-1α expressions were found on both lining and sublining area of synovium.HIF-1α expression in synovium increased gradually with the prolonged disease course.Synovial HIF-1α expression correlated significantly with arthritis activity indexes,total pathological score,synovial hyperplasia score and angiogenesis score,but not with inflammation score.CONCLUSION: HIF-1α may play important roles in the pathogenesis of RA through inducing synovial hyperplasia and angiogenesis.     

Anti-Sonic hedgehog blocking antibody enhances killing effect of PBMCs on cervical carcinoma HeLa cells

AIM: To investigate the effect of anti-Sonic hedgehog(Shh) blocking antibody on the killing effect of peripheral blood mononuclear cells(PBMCs) on cervical carcinoma HeLa cells. METHODS: The expression levels of Shh and Shh signaling molecules in HeLa cells were detected by immunocytochemistry and RT-PCR. PBMCs from health peoples were isolated by the method of Ficoll density gradient centrifugation, and then co-cultured with HeLa cells in vitro. The expression of CD3, CD69 and CD71 was assayed by flew cytometry. The concentrations of IFN-γ, IL-10 and IL-4 in culture supernatants were detected by ELISA. The killing effect of PBMCs on HeLa cells was observed under microscope. RESULTS: Shh and Shh signaling molecules were expressed in HeLa cells. The level of Shh expression didn't change significantly in the 6th passage of HeLa cells. CD3⁺ cells were increased in the co-culture system. The expression of CD69 and CD 71, and the secretion of IFN-γ were increased, while the secretion of IL-10 was decreased in the co-culture system treated with anti-Shh blocking antibody. Anti-Shh blocking antibody has no effect on the secretion of IL-4. The killing effect of PBMCs on HeLa cells was strengthened by anti-Shh blocking antibody. CONCLUSION: Anti-Shh blocking antibody promotes the activation of PBMCs and enhances the killing effect of PBMCs on cervical carcinoma HeLa cells.     

Galectin-9 regulates SHH signaling pathway to influence apoptosis of colorectal cancer HT29 cells

AIM: To investigate the effect of galectin-9 on the apoptosis of colorectal cancer HT29 cells. METHODS: Galectin-9 over-expression vector (pcDNA3.1-Galectin-9) or control vector (pcDNA3.1) was transfected into the HT29 cells. The galectin-9 over-expression was detected by real-time PCR and Western blot. Annexin V-FITC/PI double staining was used to detect the apoptosis. The protein level of activated caspase-3 and the expression of SHH signaling pathway-related proteins Smo, Gli1 and SHH in the HT29 cells were determined by Western blot. SHH signaling pathway specific inhibitor cyclopamine was used to treat the HT29 cells with up-regulated galectin-9 expression, and the apoptosis, the protein level of cleaved caspase-3 and the expression of SHH signaling pathway-related proteins Smo, Gli1 and SHH in the HT29 cells were detected by the above methods. RESULTS: Transfection with pcDNA3.1-Galectin-9 up-regulated galectin-9 expression at mRNA and protein levels in the colorectal cancer HT29 cells (P<0.05). The apoptotic rate of the HT29 cells was increased after galectin-9 up-regulation. The protein level of cleaved caspase-3 in the cells was increased, while the expression levels of SHH signaling pathway-related proteins Smo, Gli1 and SHH were decreased. Cyclopamine treatment further induced the apoptosis of the HT29 cells with up-regulation of galectin-9, increased the protein le-vels of cleaved caspase-3, and decreased the activation level of SHH signaling pathway in the HT29 cells (P<0.05). CONCLUSION: Galectin-9 induces the apoptosis of colorectal cancer HT29 cells by inhibiting SHH signaling pathway.     

Relationship between anti-CCP antibody and migration or invasion ability of fibroblast-like synoviocytes from rheumatoid arthritis patients

AIM:To investigate the relationship between anti-cyclic citrullinated peptide (anti-CCP) antibody and migration or invasion ability of fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS:FLS was isolated from 22 patients with active RA, 12 patients with osteoarthritis (OA) and 6 patients with joint injuries without arthritis history. The migration and invasion abilities were assessed by Transwell assay. RESULTS:Among the patients of RA, OA and normal controls, the numbers of migratory cells were (29.33±10.93), (9.28±7.87) and (7.00±4.07)/field, respectively, and the numbers of invasive cells were (14.35±7.67), (3.96±4.37) and (3.08±1.03)/field, respectively. The numbers of migratory and invasive cells were higher in RA than those in the other groups. The migration and invasion abilities of RA FLS from anti-CCP antibody-positive RA patients were significantly increased as compared with anti-CCP antibody-negative RA patients. Correlation analysis revealed that the migration and invasion abilities of RA FLS were correlated with the positive and high titer of anti-CCP antibody.CONCLUSION: The migration and invasion abilities of FLS from anti-CCP antibody-positive RA patients are increased. Anti-CCP antibody is possibly correlated with the increased migration and invasion abilities of RA FLS.     

Thromboxane A2 receptor induces proliferation of rheumatoid arthritis synovial cells by up-regulation of cyclooxygenase-2

AIM：To examine the effects of thromboxane A2 receptor (TXA2R), the downstream product of cyclooxygenase-2 (COX-2), on the proliferative ability and COX-2 expression in rheumatoid arthritis (RA) synovial cells. METHODS：The effects of TXA2R antagonist SQ29548 and agonist U46619 on the proliferation of RA synovial cell line MH7A were detected by MTS cell proliferation assay, and their effects on COX-2 mRNA expression in MH7A cells were also examined by real-time PCR. In addition, the possible effect of U46619 on the proliferation of MH7A cells, when COX-2 was knocked down by siRNA, was determined by BrdU cell proliferation assay. RESULTS：SQ29548 inhibited the cell proliferation and the mRNA level of COX-2 while U46619 enhanced them. Moreover, U46619 reconstitute the proliferative ability of MH7A cells to some extent that inhibited by COX-2 siRNA. CONCLUSION：In RA synovial cells, TXA2R is able to control COX-2 expression, while it also mediates the effects of COX-2, suggesting that TXA2R might be an ideal candidate for RA treatment.     

Rosiglitazone inhibits osteoclastogenesis in rheumatoid arthritis by down-regulating RANKL expression and suppressing ERK phosphorylation

AIM: To investigate the effects of rosiglitazone on fibroblast-like synoviocyte (FLS)-induced osteoclastogenesis in rheumatoid arthritis (RA) and the related mechanism. METHODS: RA-FLS were cocultured with peripheral blood monocytes from healthy volunteers in the presence of macrophage colony-stimulating factor (M-CSF) and rosiglitazone. Osteoclasts were assayed by tartrate-resistant acid phosphatase (TRAP) staining. Resorption lacunae area was identified by toluidine blue staining and quantified by image analysis software. The mRNA expression of RANKL and OPG was evaluated by real-time PCR, and the protein levels of RANKL, OPG, p-ERK, p-p38 and p-JNK were measured by Western blot. RESULTS: Compared with control group (without rosiglitazone treatment), rosiglitazone at concentration of 15 μmol/L significantly decreased the number of osteoclasts (P<0.01) and resorption lacunae area (P<0.05). The expression of RANKL at mRNA and protein levels was significantly down-regulated by rosiglitazone at concentration of 15 μmol/L, while the mRNA and protein expression of OPG was up-regulated (P<0.01). Rosiglitazone (15 μmol/L) significantly decreased the protein level of p-ERK (P<0.05), but not the protein level of p-p38 or p-JNK (P>0.05). CONCLUSION: Rosiglitazone inhibits RA-FLS-induced osteoclast formation and its resorption activity by down-regulating RANKL expression and ERK phosphorylation, suggesting that rosiglitazone may inhibit RA osteoclastogenesis and bone resorption.     

Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells

AIM: To investigate the effect of plumbagin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the apoptosis of leukemic Kasumi-1 cells. METHODS: Kasumi-1 cells were treated with plumbagin alone, recombinant soluble TRAIL(rsTRAIL) alone or the combination of plumbagin with rsTRAIL to induce apoptosis. The cell proliferation was analyzed by CCK-8 assay. Apoptosis was determined by flow cytometry with AnnexinⅤ/PI double staining and TUNEL staining. The expression of DR4 and DR5 at mRNA level was measured by real-time PCR. The expression of signal transduction proteins, such as DR5, caspase-3, caspase-8, caspasep-9, Bid, Bax and c-FLIP was detected by Western blotting. RESULTS: Both rsTRAIL and plumbagin induced the apoptosis in Kasumi-1 cells, and combination of plumbagin with rsTRAIL enhanced the apoptosis. The ratios of Annexin V-positive Kasumi-1 cells were (27.7±2.9)%, (25.6±3.1)% and (52.1±3.3)% in 100 μg/L rsTRAIL group, 2 μmol/L plumbagin group and the combination group, respectively, and the positive rate in combination group was significantly higher than those in other 2 groups. TUNEL assay demonstrated that the number of apoptotic cells in combination group was higher than that in the cells treated with rsTRAIL or plumbagin alone. Plumbagin up-regulated the expression of DR5 at mRNA level in Kasumi-1 cells, and up-regulation of DR5, activation of caspase-8 and down-regulation of c-FLIP at protein level were detected in the cells treated with plumbagin alone and the combination of plumbagin with rsTRAIL. CONCLUSION: Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells by up-regulating DR5, activating caspase-8 and down-regulating c-FLIP.     

Studies on the role of ET-1 and CGRP in the pathophysiology of ankylosing spondylitis and rheumatoid arthritis

AIM: To clarify the role of endothelin-1 (ET-1) and calcitonin gene related peptide(CGRP) in the pathophysiology of ankylosing spondylitis and rheumatoid arthritis. METHOD: Plasma/synovial fluid ET-1 and CGRP were measured by radioimmunoassay in patients with ankylosing spondylitis (AS) or rheumatoid arthritis (RA) and healthy control. RESULTS: ET-1 level in plasma of patients with AS and RA were significantly higher than that of healthy controls (P<0.01). No difference was found in plasma CGRP level between AS or RA and healthy control (P>0.05). CGRP level in synovial fluid was significantly higher than that in plasma (P<0.01), but ET-1 level was significantly lower than in plasma (P<0.01). CONCLUSION: These results suggest that ET-1 and CGRP play a pathogenic role in AS and RA.     

Expression of Hedgehog signaling molecules in rat livers after partial hepatectomy

AIM: To detect the expression of Hedgehog signaling molecules in rat livers after partial hepatectomy. METHODS: The model of rat partial hepatectomy was established by resecting the middle and left lobes of the liver. Eighteen male Sprague-Dawley rats were divided into 3 groups: sham operation group (group A), partial hepatectomy group 1 (group B) and partial hepatectomy group 2 (group C). Hepatic tissues were collected 24 h after the operation in group A and group B, and 48 h after the operation in group C. The expression of Ki-67,Sonic Hedgehog(Shh),Indian Hedgehog(Ihh) and Glioblastoma-2(Gli-2) in the hepatic tissues were detected by immunohistochemical staining and Western blotting. RESULTS: The edema and spotty necrosis in the hepatic tissues were observed in group B and group C by HE staining. The cells of different dividing stages were found in the hepatic tissues of group C. Compared with group A, the expression of Ki-67, Shh, Ihh and Gli-2 in group B (P<0.01) and group C (P<0.01) was significantly elevated, and the expression levels in group C were higher than those in group B (P<0.01). CONCLUSION: Hedgehog signaling in rat livers may be activated after partial hepatectomy and stimulate liver regeneration.     

Generation,identification and differentiation of primary ovarian insufficiency patient-derived iPSCs

AIM: To generate and identify primary ovarian insufficiency (POI) patient-derived induced pluripotent stem cells (iPSCs) and to explore their differentiation potential to primordial germ cells. METHODS: Plasmid pEB-C5 expressing reprogramming factors Oct4, Sox2, c-Myc, Klf4 and Lin28, and plasmid pEB-Tg expressing SV40 T antigen, were transfected into peripheral blood mononuclear cells (PBMCs) derived from POI patients at the same time. PBMCs were reprogrammed into iPSCs, and the pluripotency of the cells was identified. After supplementation of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4 (BMP4), the mRNA and protein expression of primordial germ cell markers was detected by real-time PCR and Western blot, respectively. RESULTS: iPSCs derived from the PBMCs of POI patient differentiated into 3-germ layer cells and maintained pluripotency by the detection of alkaline phosphatase staining, immunofluorescence, embryoid body and teratoma formation. After addition of TGF-β1 and BMP4, the primordial germ cell markers, including stem cell growth factor receptor (c-Kit), developmental pluripotency-associated 3 (STELLA/DPPA3) and DEAD box polypeptide 4 (VASA/DDX4) were increased at mRNA level (P<0.05), and VASA/DDX4 was also up-regulated at protein level in induced group. CONCLUSION: PBMCs of POI patient are reprogrammed into integration-free iPSCs in vitro and maintain pluripotency. They differentiate into primordial germ cells by adding TGF-β1 and BMP4.     

Effect of Hedgehog signaling pathway inhibitor on biological behavior of intrahepatic cholangiocarcinoma cell line RBE

AIM: To investigate the effect of cyclopamine, a Hedgehog(Hh) signaling pathway inhibitor, on the biological behavior of intrahepatic cholangiocarcinoma cell line RBE. METHODS: The proliferation of RBE cells was detected by cell counting with Typan blue staining and MTT assay, and the apoptosis was analyzed by the flow cytometry. The Transwell invasive cabin assay was used to detect the invasion ability, and Western blot was used to determine the protein expression of Gli1 and MMP-9 in the RBE cells before and after cyclopamine treatment. RESULTS: Cyclopamine inhibited the growth of RBE cells in a time-and dose-dependent manner. After cyclopamine treatment for 24 h, 48 h and 72 h, the apoptotic rates were significantly higher than those in control group. In control group, the number of cells invading through the Matrigel of invasion chamber was 154.52±13.61, while in experimental group it was 62.00±12.17, indicating that the invasion ability of the cells declined significantly. Furthermore, Western blot showed that the protein levels of Glil and MMP-9 in the RBE cells were decreased after treatment with cyclopamine for 24 h and 48 h. CONCLUSION: Blockage of the Hh signaling pathway with cyclopamine suppresses the proliferation, promotes the apoptosis and inhibits the invasion ability of RBE cells.