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1.
AIM:To compare the cDNA sequences of ASBT (apical Na+/bile acid cotransporter) gene cloned from human kidney and intestine tissues, and to determine subcellular localization of ASBT in renal tubular epithelial cells and expression site of ASBT in human kidney tissue.METHODS:The total RNA was extracted from human kidney and intestine tissues. The full-length cDNA gene of ASBT was amplified by RT-PCR technique using primers with 8-peptide FLAG tag, sequenced and inserted into eukaryotic expression vector to construct ASBT protein expression vector, which was then transfected into swine renal tubular epithelial cell line LLC-PK1. The subcellular localization of ASBT protein was determined by immunofluorescence staining and laser confocal microscope. Immunohistochemical analysis was used to observe expression position of ASBT in kidney tissue.RESULTS:The results revealed that the sequence of kidney ASBT cDNA gene was identical to that of human intestine ASBT gene. Western blotting analysis indicated that ASBT protein was correctly expressed in LLC-PK1 cells. Confocal scanning analysis showed that ASBT protein was mainly localized at the cellular plasma membrane, consistent with the predicting result obtained by bioinformatics. The results of immunohistochemistry revealed that ASBT was expressed at brush border membrane of proximal renal tubular cells, but not expressed in distal tubule and renal interstitium.CONCLUSION:The gene sequence of kidney ASBT is the same as that of intestine ASBT and ASBT protein is expressed at the lumen (apical) membrane of proximal renal tubular epithelial cells.  相似文献   

2.
AIM: To express and purify human histydyl-tRNA synthetase analogue and prepare its polyclonal antibody. METHODS: The open reading frame of the synthetase gene was amplified by PCR, then recombined into prokaryotic expression vector pQE30 and transformed into E.coli M15 for expression. Rabbits were immunized with purified expression products. RESULTS: Prokaryotic expression vector pQE30 of human histydyl-tRNA synthetase analogue was constructed. The recombined protein was purified by Ni affinity chromatography, and high titer polyclonal antibody was successfully prepared. CONCLUSION: Recombined prokaryotic expression vector, the purified protein and prepared polyclonal antibody were the necessary materials for further study of this protein.  相似文献   

3.
AIM:To study the expression and function of novel gene AngRem104.METHODS:Northern blot was performed to detect the distribution of AngRem104 in human multiple normal tissues as well as the effect of AngⅡ and AT1R antagonist (losartan) on AngRem104 expression. The sense and antisense eukaryotic expression vectors of AngRem104 were constructed and transfected into human mesangial cells. RT-PCR was used to detect the expression of FN when AngRem104 was over-expressed. Primary sequence and motif analysis of AngRem104 protein were performed by on-line ExPasy predictive tools.RESULTS:AngRem104 was predicted to localize at the cellular nucleus. It was widely expressed in human heart, placenta, liver, muscle, kidney and pancreas. Moreover, the up-regulated expression of AngRem104 induced by AngⅡ was inhibited by losartan in a dose-dependent manner.CONCLUSION:AngRem104 is a novel nuclear protein related to the expression of fibronectin and could be up-regulated by AngⅡ in human MC.  相似文献   

4.
AIM:To explore the effects of PMA(phorbol-12-myristate-13-acetate, a tumor promoter, mimicking the action of diacylglycerol on PKC)and laminin on the adhesion and the proliferation of human hepatocellular carcinoma cells, and provide a new clue to liver cancer treatment.METHODS:Human hepatocellular carcinoma cell line(BEL-7402)was used to identify the endogenous laminin and protein kinase C-α(PKC-α) expression, and the effects of laminin and PMA on the adhesion and the proliferation were also investigatedin vitro.RESULTS:By the effect of exogenous laminin, human hepatocellular carcinoma cell (BEL-7402) possessed endogenous laminin expression and increased the adhesion and the proliferation, which was showed the synergistic action by the effect of PMA in combination. By the action of PMA alone, the proliferation and the PKC-α expression increased by exogenous laminin were decreased, and the adhesion and the endogenous laminin expression were increased.CONCLUSIONS:The finding suggested that the adhesion and the proliferation of human hepatocellular carcinoma cell were closely related to the effects of endogenous or exogenous laminin, which were associated with cPKC-α activity. Therefore, the application of anti-laminin antibody in combination with PKC antagonist might be a new clue to find out the therapy for liver cancer.  相似文献   

5.
AIM: To study the expression of endostatin in ischemic myocardium of myocardial infarction (MI) rats in various periods and the correlation with VEGF expression and microvascular density (MVD).METHODS: Thirty-two male Sprague-Dawley rats after myocardial infarction were randomly divided into 7, 14, 21 and 28 days group.The sham group was normal control group (eight rats in each group).The expression of endostatin, VEGF and MVD in ischemic myocardium were observed by immunohistochemistry.RESULTS: The expression of endostatin significantly increased in the ischemic myocardium after MI, peaked at 7 days, then gradually decreased at 14, 21 and 28 days.The endostatin level at 28 days was the same as the shams.The changing trends of expression of endostatin in ischemic myocardium after MI were similar to that of VEGF and were significantly correlated with the MVD.CONCLUSION: The expression of endostatin increased in ischemic myocardium of myocardial infarction rats.The changing trends of endostatin were similar to that of VEGF and positively correlated with the MVD.These data suggest that endostatin may modulate ischemic myocardium angiogenesis after myocardial infarction.  相似文献   

6.
AIM: To investigate the effects of phillyrin on vascular endothelial growth factor (VEGF) and endostatin expression in lung tumor tissues isolated from Lewis lung carcinoma. METHODS: The expression of VEGF and endostatin in control individuals and the patients with lung cancer was determined by immunohistochemistry. In the animal experiment, 5 groups of animals were examined: control, tumor model, and tumor model with 3 different concentrations of phillyrin treatments. For preparation of transplanted tumor model, Lewis cells were subcutaneously injected into the right limb armpit of the nude mice. After that, phillyrin was administered via oral gavage once daily for 20 d at dose of 5 or 10 g/kg, or twice daily at 10 g/kg. Lung tumor tissues isolated from each group were observed by hematoxylin-eosin staining. VEGF and endostatin expression were examined by immunohistochemistry. RESULTS: VEGF expression was increased in lung tumor tissues as compared with normal and pericarcinous tissues, while endostatin expression was decreased. Phillyrin significantly inhibited the tumor size and tumor tissue density dose-dependently, which was accompanied with a decrease in VEGF expression and an increase in endostatin expression. CONCLUSION: Phillyrin inhibits the development of lung tumor through reducing VEGF expression and increasing endostatin expression.  相似文献   

7.
AIM: To study the inhibitory effect of the extract of Oratosquilla (EOS) on the migration and vasculogenic mimicry in human poorly differentiated nasopharyngeal carcinoma cell line CNE-2.METHODS: CNE-2 cells were cultured in the medium with different concentrations of EOS (0 mg/L, 125 mg/L, 250 mg/L and 500 mg/L). The migration of CNE-2 cells and the formation of tube-like structures (TLSs) by CNE-2 cells were determined with wound healing assay and in vitro anti-angiogenesis test, respectively. The formation of TLSs by CNE-2 cells and their structural characteristics were observed by anti-angiogenesis test on the Matrigel. The protein expression of fascin 1 and vascular endothelial growth factor (VEGF) was detected by Western blotting.RESULTS: Compared with control group, EOS significantly decreased the migration velocity of CNE-2 cells in a dose-dependent manner. CNE-2 cells formed TLSs on the Matrigel, and the formation of TLSs by CNE-2 cells was inhibited by EOS in a dose-dependent manner. The expression of fascin 1 and VEGF in CNE-2 cells was also decreased after treatment with EOS. A positive correlation between the expression of fascin 1/VEGF and the formation of TLSs by CNE-2 cells was observed.CONCLUSION: CNE-2 cells form TLSs on the Matrigel, and EOS inhibits the migration and vasculogenic mimicry of CNE-2 cells, which are related with down-regulating the expression of fascin 1 and VEGF in CNE-2 cells.  相似文献   

8.
AIM: To explore the therapeutic strategies of heat-clearing and detoxifying (HCDT) and Yin-invigorating and fluid-supplementing (YIFS), the method of Chinese medicine, on the high density lipoprotein (HDL) and alkaline phosphatase (ALP) molecules, which are associated with endotoxin-degradation. METHODS: The animal model of endotoxemia was established by the injection of E. Coli lipopolysaccharides through rabbits ear vein. The endotoxemic rabbits were treated respectively with two representative herbal preparations of therapeutic principles against febrile diseases: HCDT or YIFS preparations. The serum levels of interleukin-1 (IL-1), tumor necrosis factor α (TNF-α) and HDL, the ALP activity, the expression of ALP mRNA in liver and kidney tissues were observed. RESULTS: The serum levels of IL-1 and TNF-α in HCDT group were significantly decreased, while the serum ALP activity and the expression of ALP mRNA in liver and kidney tissues were obviously increased, as compared with those in model group. Meanwhile, the serum levels of IL-1 and TNF-α in YIFS group were significantly reduced, and its plasma HDL level was elevated. CONCLUSIONS: Both the herbal preparations, HCDT and YIFS, have the scavenging effects on the overproduction of inflammatory cytokines such as IL-1 and TNF-α induced by endotoxin, but their effects on the endotoxin-degrading molecules might be different. HCDT principally increases ALP activity and enhances ALP expression in liver and kidney tissues, while YIFS might preferably facilitate the elevation of plasma HDL level.  相似文献   

9.
AIM: To determine the expression of aquaporin 3 (AQP3) in transitional and peripheral zone of human prostate tissue.METHODS: AQP3 mRNA expression was analyzed in five prostate tissues by RT-PCR. AQP3 protein expression and localization were characterized by Western blotting and immuno-staining with polyclonal anti-AQP3 antibody.RESULTS: The results of RT-PCR and Western blotting showed that AQP3 was expressed in transitional and peripheral zone of prostate tissue. The AQP3 protein expression in transitional zone was higher than that in peripheral zone. The results of immuno-staining indicated that AQP3 protein was expressed in secretory cells of prostate.CONCLUSION: The AQP3 was expressed in prostate tissue, suggesting that AQP3 play an important role in the secretion of prostate liquid.  相似文献   

10.
AIM: To investigate the therapeutic action of secreted endostatin (ES) on breast cancer cells. METHODS: Retroviral-mediated endostatin gene was transferred to breast cancer cell line MDA-MB-231. The ES biological properties and function were evaluated by polymerase chain reaction (PCR), MTT and a murine xenograft model. RESULTS: After retroviral transduction, endostatin genetically modified breast tumor cells were confirmed by PCR, and the integration and durative expression of endostatin gene was successfully committed. Compared with controls, endostatin secreted by genetically modified cells markedly inhibited endothelial cell proliferation (P<0.05) while the influences on the growth of MDA-MB-231 cell line in vitro were not found (P>0.05). The results of the transplanted subcutaneous tumor model in nude mice suggested that the subcutaneous growth of MDA-MB-231 was significantly inhibited by the expression of endostatin gene (P<0.05). In experimental groups, the tumor microvascular density (MVD) and VEGF expression were decreased. CONCLUSION: Retroviral- mediated overexpression of endostatin inhibits the proliferation of vascular endothelial cells and angiogenesis that associated with tumor growth in vivo via the paracrine pathway, which has a potential effect in the angiostatic gene therapy for breast cancer.  相似文献   

11.
AIM: To determine the role of Fas antigen and caspase-8 in modulating apoptosis of osteosarcoma cells induced by bacterial redox protein azurin. METHODS: The human osteosarcoma cell line U2OS was treated with bacterial redox protein azurin (150 mg/L) for 6, 12, 24 and 48 h, respectively. Cell immunohistochemistry and quantitative image pattern analysis were applied for detecting the expression of Fas antigen. Caspase-8 activity was detected using caspase-8 fluorescent assay kit. The apoptotic rate was measured by FCM. RESULTS: Compared with the control group, the expression of Fas antigen and activity of caspase-8 significantly increased in U2OS cells treated with 150 mg/L azurin (P<0.01). There was positive correlation between the expression of Fas antigen and activity of caspase-8 (r=0.873, P<0.01). The increased Fas antigen expression had the function to transfer apoptotic signals and the anti-Fas antibody promoted azurin induced apoptosis through increasing Fas antigen expression. IETD-FMK blocked the activation of procaspase-8 and reduced apoptosis of U2OS cells in the presence of azurin or anti-Fas antibody. The apoptotic rates in azurin group and anti-Fas antibody group were significantly different from the inhibitor group (P<0.01). CONCLUSION: The Fas-dependent signalling is the important pathway of azurin inducing apoptosis in U2OS cells. The up-regulation of csepase-8 may play an important role.  相似文献   

12.
AIM: To investigate the expression of angiotensin-converting enzyme 2 (ACE2) in nephritic epithelium of primates. METHODS: The expression of ACE2 in Vero E6 cells was detected by the techniques of RT-PCR and immunocytochemistry (ICC) techniques. The distribution of ACE2 protein in kidney tissues of two Rhesus monkeys and two normal human cases was observed by immunohistochemistry (IHC) techniques. RESULTS: Vero E6 cells were found to express both ACE2 mRNA and protein. ACE2 protein was mainly located in epithelium of proximal tubules of Rhesus monkey and human kidney. CONCLUSION: The expression of ACE2 in epithelium of primate kidney may provide the condition for SARS-CoV entry, which may be one of the reasons for inducing renal damage in SARS patients.  相似文献   

13.
AIM: To investigate the transfection efficiency of mouse liver with non-viral vector containing manganese superoxide dismutase (Mn-SOD) gene. METHODS: The eukaryotic expression vector, gWiz/Mn-SOD, encoding human manganese superoxide dismutase was constructed. The plasmids of gWiz/Mn-SOD were mixed with cationic lipids, followed by injection into mice via branch of superior mesenteric vein, to induce Mn-SOD over-expression in murine liver detected by RT-PCR, Western blotting, SOD activity and immunohistochemical staining. RESULTS: gWiz/Mn-SOD transfection resulted in the obvious expression of exogenous Mn-SOD mRNA and protein in hepatic tissues at 8 hours after injection, and elevated mitochondria SOD activity 8.4 times in transfected hepatocytes than that in non-transfected cells at 72 hours after injection. It was showed that nearly 70% of mouse hepatocytes was obviously Mn-SOD positive after transfection. CONCLUSION: High expression efficiency of Mn-SOD gene in mouse liver is achieved safely, by injection of gWiz/Mn-SOD and cationic lipid mixture into branch of superior mesenteric vein.  相似文献   

14.
AIM:To express recombinant hCD154-GST fusion protein, to prepare anti-hCD154 monoclonal antibody, and to investigate the effect of anti-hCD154 monoclonal antibody on graft rejection. METHODS AND RESULTS: Total RNA was prepared from human peripheral blood mononuclear cell (PBMC) activated with 10ng/mL PMA and 1 μg/mL PHA for 8h, the total RNA was reversetranscribed to cDNA. The entire coding region and a part of the 3'non-coding regions were amplified by PCR using a pair of primers designed and synthesized according to the sequence of human CD154 gene from gene bank. The amplified product, a 820bp DNA fragment was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST). The cloned insert was identified by double digestion of the cloned pGEX-4T-1 plasmid with retriction enzymes BamHⅠand EcoRⅠ.The fusion protein expression plasmid of PGEX-4T-1/hCD154 was constructed, then transformed to E coli BL21. The human CD154-GST fusion protein expression was induced by IPTG in BL21. The expression of recombinant 26kD GST and 55kD human CD154-GST fusion protein were confirmed by SDS-PAGE. CONCLUSION: We have express the recombinant human CD154-GST fusion protein. The expressed hCD154-GST fusion protein will be used to prepare anti-hCD154 monoclonal antibody, to investigate the role of anti-CD154 monoclonal antibody on graft rejection.  相似文献   

15.
AIM: To study the expression of connective tissue growth factor (CTGF) induced by hypoxia, and the role and mechanism of hypoxia on promoting renal interstitial fibrosis. METHODS: Renal interstitial fibrosis was induced by unilateral ureteral obstruction (UUO) in rat animal model for 9 days as in vivo studies; marker of hypoxia-HIF-1α mRNA and protein, the expression of CTGF in the obstructed kidneys were assessed by RT-PCR, immunohistochemistry and Western blotting respectively. In vitro, normal rat kidney interstitial fibroblast cells (NRK-49F) were exposed to hypoxia (1%O2) for up to 6 hours, hypoxia was confirmed by detecting the expression of HIF-1α protein in cells, cellular level of CTGF mRNA and protein were assessed by RT-PCR and Western blotting respectively. RESULTS: Neither HIF-1α mRNA nor HIF-1α protein was expressed in the kidney from sham-operated group of rats. High level of HIF-1α mRNA were occurred, and strongly HIF-1α positive immunostaining were seen in the tubular and interstitial cells in kidney from UUO rats. Expression and location of CTGF protein were paralleled and relevant with the expression of HIF-1α protein in kidney of UUO rats. In cultured NRK-49F cell line, subjected to hypoxia even for 6 hours stimulated the expression of CTGF mRNA and protein. CONCLUSION: Our results indicated that hypoxia could stimulate the expression of CTGF mRNA and protein in kidney from UUO rats, which may in turn contribute to renal interstitial fibrosis.  相似文献   

16.
AIM: To investigate the distribution of endogenous sulfur dioxide (SO2) and its generating enzyme (glutamate oxaloacetate transaminase, GOT) in normal rats. METHODS: Wistar rats were anaesthetized and heart, liver, lung, kidney and vascular tissues (including aorta and pulmonary, mesenteric, kidney and tail arteries) were isolated rapidly. SO2 content was determined by reverse-phase HPLC with fluorescence detection and GOT was detected in various tissues by quantitative real-time RT-PCR and enzymatic methods. GOT mRNA expression in arteries was studied through in situ hybridization. RESULTS: SO2 was endogenously generated in various rat tissues, including heart, liver, lung, kidney and arteries. The SO2 content was higher in arteries than that in other tissues. GOT enzyme and GOT mRNA expression were higher in heart, liver and kidney than those in arteries, and GOT1/GOT2 mRNA located in endothelia and vascular smooth muscle cells near endothelial layer. CONCLUSION: SO2 is endogenously generated in cardiovascular system, and the SO2 content in arteries is higher than that in other organs. GOT enzyme and GOT mRNA expression are low in arteries, and this phenomenon may be related with local metabolism.  相似文献   

17.
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18.
AIM: To approach the changes of endostatin levels in BALB/c nude mice bearing human nasopharyngeal carcinoma(NPC) in different period (5, 10, 20, 30 and 40 days) and the relationship between endostatin and tumor's development. METHODS: BALB/c nude mice bearing NPC was reproduced by hypodermic implantation of human CNE-2 cells into right-side of axillary fossa. The level of plasma endostation was detected, and the weight of isolated tumors was measured. On the basis of the regulation of these changes, their relationships were explored. RESULTS: At 5 days [(137.61±53.41) μg/L] or 10 days [(103.06±17.33) μg/L] endostatin level had no apparent alternation in comparison with control group [(113.56±21.74) μg/L, P>0.05]. At 20, 30 and 40 days concentration of endostatin[(212.80±85.91) μg/L,(293.63±62.53) μg/L, (271.57±32.45) μg/L, respectively] were higher than that of the control group (P<0.05). Along with the development of the tumors, both the levels of endostatin and tumors weight increased. There was a positive correlation between the level of endostatin and tumor weight (r=0.687, P<0.05). CONCLUSION:These results suggested that endostatin links with the development of NPC.  相似文献   

19.
AIM: To study the expression of eukaryotic elongation factor 1A2 (eEF1A2) in the hepatocellular carcinoma (HCC) tissues and the effects of eEF1A2 over-expression on the biological behaviors of the HCC cells. ME-THODS: The expression of eEF1A2 at mRNA and protein levels in the HCC tissues and matched liver tissues from 62 HCC patients, and 20 normal liver tissues were detected by the methods of real-time PCR and immunohistochemical staining, respectively. The mRNA and protein expression of eEF1A2 in the HCC cells was also determined by real-time PCR and Western blot, respectively. The lentivirus containing eEF1A2 gene was constructed, and was used to infect the HCC cells with low eEF1A2 expression. The expression of eEF1A2 at mRNA and protein levels in the infected cells was detected by real-time PCR and Western blot, respectively. The cell activity, cell cycle and mRNA expression of albumin were measured by MTT assay, DNA ploid analysis and real-time PCR, respectively.RESULTS: The mRNA expression levels and protein expression positive rates of eEF1A2 in the 62 cases of HCC tissues, were significantly higher than those of 62 matched liver tissues and 20 normal liver tissues (P<0.01). eEF1A2 mRNA and protein were highly expressed in SMMC-7721 cells and BEL-7402 cells, and expressed in SK-HEP-1 cells at low level. The expression of eEF1A2 at mRNA and protein levels in the SK-HEP-1 cells was significantly enhanced by infection of GV287-eEF1A2 expression lentivirus.Compared with negative control group (transfected with negative control lentivirus), the cell activity in eEF1A2 over-expression group (transfected with GV287-eEF1A2 expression lentivirus) was significantly enhanced, the mRNA expression of albumin was remarkably reduced, and the cells in G0/G1 phase were significantly decreased with increased percentage of the cells in S and G2/M phases.CONCLUSION: eEF1A2 is selectively over-expressed in human HCC cancer tissues. eEF1A2 might be a putative oncoprotein in HCC. eEF1A2 over-expression has noticeable effects on the HCC cell proliferation enhancement, differentiation inhibition, and cell cycle acceleration through the G0/G1 phase to S phase and G2/M phases.  相似文献   

20.
AIM: To investigate the preventive effect and mechanism of anti-insulin-like growth factor binding protein related protein 1(IGFBPrP1) antibody on hepatic fibrosis induced by thioacetamide (TAA) in mice.METHODS: Twenty-four male C57BL/6 wild-type mice were randomly divided into 3 groups (n= 8 in each group): normal control group, TAA group (4 weeks) and TAA+anti-IGFBPrP1 antibody group (4 weeks). The morphological changes of liver tissues were observed. The expression levels of α-smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β1), Smad3, phosphorylated Smad2/3 (p-Smad2/3), fibronectin (FN), collagen I, collagen Ⅲ and IGFBPrP1 were detected by the methods of immunohistochemistry and Western blotting.RESULTS: In TAA group (4 weeks), obvious injury of liver was observed, and the expression levels of α-SMA, TGF-β1, Smad3, p-Smad2/3, FN, collagen Ⅰ, collagen Ⅲ and IGFBPrP1 were significantly increased as compared with normal control group (P<0.01). Compared with TAA group (4 weeks), the injury of the liver was alleviated and the expression levels of the proteins above were decreased in TAA+anti-IGFBPrP1 antibody group (4 weeks, P<0.01). IGFBPrP1 was positively correlated with TGF-β1, Smad3, p-Smad2/3, FN and collagen I (P<0.01). CONCLUSION: Anti-IGFBPrP1 antibody prevents TAA-induced hepatic fibrosis in mice by inhibiting the activation of hepatic stellate cells, reducing the expression of p-Smad2/3 and inhibiting the TGF-β1/ Smad3 signal transduction, thereby depressing the deposition of extracellular matrix in liver tissues.  相似文献   

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