The relation between the expression and activity of MMP-9 in C-reactive protein (CRP)-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B (NF-κB) was studied to investigate the possible role of CRP in plaque destabilization. Human THP-1 cells were incubated in the presence of CRP at 0 (control group), 25, 50 and 100 μg/mL (CRP groups) for 24 h. In PDTC (a specific NF-κB inhibitor) group, the cells were pre-treated with PDTC at 10 μmol/L and then with 100 μg/mL CRP. The conditioned media (CM) and human THP-1 cells in different groups were harvested. MMP-9 expression in CM and human THP-1 cells was measured by ELISA and Western blotting. MMP-9 activity was assessed by fluorogenic substrates. The expression of NF-κB inhibitor α (IκB-α) and NF-κB P⁶⁵ was detected by Western blotting and ELISA respectively. The results showed that CRP increased the expression and activity of MMP-9 in a dose-dependent manner in the human THP-1 cells. Western blotting revealed that IκB-α expression was decreased in the cells with the concentrations of CRP and ELISA demonstrated that NF-κB P65 expression in the CRP-induced cells was increased. After pre-treatment of the cells with PDTC at 10 μmol/L, the decrease in IκB-α expression and the increase in NF-κB P⁶⁵ expression in the CRP-induced cells were inhibited, and the expression and activity of MMP-9 were lowered too. It is concluded that increased expression and activity of MMP-9 in CRP-induced human THP-1 cells may be associated with activation of NF-κB. Down-regulation of the expression and activity of MMP-9 may be a new treatment alternative for plaque stabilization by inhibiting the NF-κB activation.

The roles of voltage-dependent K⁺ channels during activation and damage in alveolar macrophages (AMs) exposed to different silica particles were examined. Rat AMs were collected by means of bronchoalveolar lavage, and were adjusted to 5×10⁵/mL. After AMs were exposed to different concentrations (0, 25, 50, 100, 200 μg/mL) of quartz particles and 100 μg/mL amorphous silica particles for 24 h, the voltage-depended K⁺ current in AMs was measured by using patch clamp technique. Meanwhile the leakage of lactate dehydrogenase (LDH) and the viability of AMs were detected respectively. Patch clamp studies demonstrated that AMs possessed outward delayed and inward rectifying K⁺ current. Exposure to quartz particles increased the outward delayed K⁺ current but it had no effect on inward rectifier K⁺ current in AMs. Neither of the two K⁺ channels in AMs was affected by amorphous silica particles. Cytotoxicity test showed that both silica particles could damage AM membrane and result in significant leakage of LDH (P<0.05). MTT studies, however, showed that only quartz particles reduced viability of AMs (P<0.05). It is concluded that quartz particles can activate the outward delayed K⁺ channel in AMs, which may act as an activating signal in AMs to initiate an inflammatory response during damage and necrosis in AMs induced by exposure to quartz particle. K⁺ channels do not contribute to the membrane damage of AMs.

This study investigated the ability of millimeter-wave (MMW) to promote the differentiation of bone marrow stromal cells (BMSCs) into cells with a neural phenotype. The BMSCs were primarily cultured. At passage 3, the cells were induced by β-mercaptoethanol (BME) in combination with MMW or BME alone. The expressions of nucleostemin (NS) and neuron-specific enolase (NSE) were detected by immunofluorescent staining and Western blotting respectively to identify the differentiation. The untreated BMSCs predominately expressed NS. After induced by BME and MMW, the BMSCs exhibited a dramatic decrease in NS expression and increase in NSE expression. The differentiation rate of the cells treated with BME and MMW in combination was significantly higher than that of the cells treated with BME alone (P<0.05). It was concluded that MMW exposure enhanced the inducing effect of BME on the differentiation of BMSCs into cells with a neural phenotype.

A protocol for the isolation, purification and culture of motor neurons from newborn rat spinal cord was described and the effect of glial cell line-derived neurotrophic factor (GDNF) on the growth of neurite of motor neurons was investigated in vitro. Spinal motor neurons (SMNs) were dissociated from ventral spinal cord of postnatal day 1 rats. The culture system for SMNs was established by density gradient centrifugation, differential adhesion, and use of serum-free defined media and addition of exogenous GDNF. After 72-h culture, the cells displayed the characteristic morphology of motor neurons, exhibited extensive neuritic processes and were positive for choline acetyltransferase (ChAT) expression. The neurite length of SMNs in GDNF groups was significantly longer than that in control group (P<0.05). This protocol can be adapted for various postnatal motor neurons studies.

Eukaryotic expression vectors carrying the small hairpin RNA (shRNA) for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside (PAN)-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN treatment+shRNA transfection group, and PAN treatment+ negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.

The effects of stromal-derived factor 1 preconditioning (PC) on apoptosis of bone mesenchymal stem cells (BMSCs) treated with hypoxia plus serum deprivation were investigated. Bone mesenchymal stem cells were cultured with the whole marrow-adherence technique. RT-PCR and immunohistochemistry were used to detect the expression of CXCR4. BMSCs were incubated in medium for 24 h with 10 ng/mL and 100 ng/mL SDF-1 respectively, and then they were treated with hypoxia plus serum deprivation for 6 h. Apoptosis rate was determined by flow cytometry and TUNEL method. The results showed that BMSCs had CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group as compared with the control group, and 100 ng/mL SDF-1 PC group had the lowest level of apoptosis. It was concluded that SDF-1 preconditioning suppresses the apoptosis of BMSCs treated with hypoxia plus serum deprivation.

This investigation describes a new precise, sensitive and accurate stereoselective RP-HPLC method for determination of the enantiomers of a novel α- and β-receptor blocking agent, 1-[4-(2-methoxyethyl) phenoxy]-3-[[2-(2- methoxyphenoxy) ethyl]amino]-2-propanol (TJ0711), in rat plasma. GITC was used for precolumn derivatization of TJ0711 enantiomers. Enantiomeric resolution was achieved on a Eurospher-100 C18 column (250 mm×4.6 mm ID, 5-μm particle size), with UV detection at 255 nm, and the mobile phase consisted of acetonitrile and water (58:42, v/v) containing 0.02% glacial acetic acid (v/v). Using the chromatographic conditions described, TJ0711 enantiomers were well resolved with mean retention time of 10.2 and 11.5 min, respectively. Linear response (r>0.999) was observed over the range of 0.125–12.5 μg/mL of TJ0711 hydrochloride enantiomers. The mean relative standard deviation (RSD%) of the results of within-day precision was ⩽ 10%. The proposed method was found to be suitable and accurate for the quantitative determination of TJ0711 enantiomers in rat plasma, and it can be used in pharmacokinetic studies.

The current study was designed to determine the safety, tolerability and pharmacokinetic parameters of recombinant human parathyroid hormone [rhPTH (1–84)] used for the treatment of osteoporosis. In the single-dose format pharmacokinetic study, thirty-six healthy male volunteers received three dose levels of rhPTH (1–84) subcutaneously: 1, 2, and 4 μg/kg. The blood was timing drawn and the serum concentration of rhPTH (1–84) was determined by enzyme linked immunosorbent assay (ELISA). Serum concentration-time curves of PTH (1–84) exhibited a double-peak pattern, the first peak appearing about 10 to 30 min after administration and the second peak occurring about 1.5 to2 h after administration. Serum terminal half-time of PTH (1–84) was approximately 2 h. The parameters indicated the serum levels were directly proportional to the administered dose, with the mean C_max and AUC_0–24 ranging from approximately 543.47 to 1845 pg/mL and 2358.6 to 9232.12 pg·h·mL⁻¹ over the dose range. The drug was well tolerated, the clinical symptoms were generally mild and of short duration.

Helicobacter pylori (H. pylori) infection causes peptic and duodenal diseases in humans. Among a 32-protein family of outer membrane proteins, a porin-like protein, HopE, has been a subject of note, mainly for its conservative nature among H. pylori, and for its potential as a vaccine candidate. To achieve stable surface expression of this host cell-toxic protein, hopE gene was introduced into pBAD expression system. After induction with arabinose, all 15 randomly-chosen E. coli LMG 194 colonies from 3 successive passages could express HopE protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indirect immunofluorescence confirmed the expression of HopE on E. coli cell surface.

The roles of NF-kappaB (NF-κB) expression, Bax activity and cytochrome C (Cyt C) release, apoptosis of islet cells induced by high concentration glucose were explored in vitro. Pancreatic islet cells, which were isolated from Kunming mice, were cultured with different concentrations of glucose in DMEM, and divided into the following groups: G1, G2, G3, G4, G5, and G6 groups, corresponding to the glucose concentrations of 5.6, 7.8, 11.1, 16.7, 22.5, and 27.6 mmol/L, respectively. After culture for 120 h, insulin secretion was evaluated by radioimmunoassay, and the NF-κB expression was detected by immunocytochemistry. Bax activity and Cyt C release were measured by immunofluorescence, and apoptosis was examined by Hoechst33342 assay. The results showed that in G1, G2 and G3 groups, insulin secretion was enhanced with the increase of glucose concentration, and the NF-κB expression was also increased (P<0.05), but Bax activity, Cyt C release and apoptosis rate showed no significant difference among them. However, in G4, G5, and G6 groups, apoptosis rate of islet cells, NF-κB expression, Bax activity, and Cyt C release were all significantly increased, and insulin secretion was impaired as compared with G1, G2, and G3 groups (P<0.05). It was concluded that the exposure of islet cells to high glucose could induce islet cells apoptosis as well as impaired insulin secretion. The NF-κB signaling pathway and mitochondria pathway in islet cells might play some roles in the progressive loss of islet cells in diabetes. The inhibition of the NF-κB expression could be an effective strategy for protecting pancreatic islet cells.

Histone deacetylase was overexpressed in a variety of cancers and was closely correlated with oncogenic factors. The histone deacetylase inhibitor, trichostatin A (TSA) was shown to induce apoptosis in many cancer cells. However, the mechanism of TSA on induction of cancer cells apoptosis is poorly understood. This study was designed to characterize the global gene expression profiles before and after treatment of human leukemia cell line Molt-4 with TSA. Flow cytometry, MTT and DNA ladder were used to observe the effect of TSA on the apoptosis of MOLT-4 cells and normal human peripheral blood mononuclear cells (PBMC). Microarray, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the difference of gene and protein expressions of Molt-4 cells after incubation of the cells with TSA. The results showed that TSA could induce Molt-4 apoptosis in dose- and time-dependent manners but spared PBMCs. Microarray analysis showed that after incubation with TSA for 9 h, 310 genes were upregulated and 313 genes were deregulated. These genes regulate the growth, differentiation and survival of cells. Among these genes, STAT5A was down-regulated by 80.4% and MYC was down-regulated by 77.3%. It was concluded that TSA has definite growth-inhibiting and apoptosis-inducing effects on Molt-4 cells in time- and dose-dependent manners, with weak cytotoxic effects on PBMCs at the same time. The mechanism of TSA selectively inducing apoptosis and inhibiting growth may be ascribed to the changes of pro-proliferation genes and anti-apoptosis genes.

The inhibitory effect of wortmannin on leukemic cells and the possible mechanisms were examined. K562 cells were treated with wortmannin of various concentrations (3.125–100 nmol/L) for 0–72 h. MTT assay was used to evaluate the inhibitory effect of wortmannin on the growth of K562 cells. Cell apoptosis was detected by both Annexin-V FITC/PI double-labeled cytometry and transmission electron microscopy (TEM). The expression of p-Akt, T-p-Akt, NF-κBp65 and IKK-κB was determined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that wortmannin obviously inhibited growth and induced apoptosis of K562 cells in vitro in a time- and dose-dependent manner. The IC₅₀ value of wortmannin for 24 h was 25±0.14 nmol/L. Moreover, wortmannin induced K562 cells apoptosis in a dose-dependent manner. TEM revealed typical morphological changes of apoptosis in wortmannin-treated K562 cells, such as chromatin condensation, karyopyknosis, karyorhexis and apoptotic bodies. Additionally, several important intracellular protein kinases such as p-Akt, NF-κBp65 and IKK-κB experienced degradation of various degrees in a dose-dependent manner both at protein level and transcription level when cultured with wortmannin, but the expression of total Akt showed no change. It is concluded that wortmannin can inhibit the proliferation and induce apoptosis of K562 leukemia cells possibly by down-regulating the survival signaling pathways (PI3K/Akt and NF-κB channels).

In the ApoE^−/− mouse model of atherosclerosis (AS) stable plaque, the expression and location of intracellular tissue factor (TF) in the cellular components of AS stable plaque were investigated in order to explore the cellular mechanism of AS thrombosis. Pathological changes of the stable plaque were observed under a microscope. The expression of TF protein was examined in aortic stable plaque of mice by using immunohistochemistry. Color image planimetric system was used to analyze the histological components of the stable plaque and the TF distribution. Under the confocal microscope, the intracellular TF location in the stable plaque of mice was observed. The results showed the cellular area was the major part of stable plaque (67.36%±6.52%, P<0.01). The percentage of total area occupied by cellular area was significantly larger than atheromatous gruel and acellular area (P<0.01). Macrophages and smooth muscle cells (SMC) were major cells in the cellular area. The percentage of total area occupied by SMC was significantly larger than by macrophages (P<0.01). Multiple linear regression analysis showed there was a positive correlation between TF area and SMC area (r=0.616, P=0.008), and no correlation was found between TF area and macrophage area (r=0.437, P=0.08). Pictures of color image planimetric analysis of TF and SMC were merged to highlight areas with co-localization (yellow), it was concluded that the process could be a cell-mediated TF expression in the stable plaque. SMC may be the major source of TF in AS without plaque rupture.

To examine the relationship between the levels of the serum vascular endothelial growth factor (VEGF) and the micrometastasis of peripheral blood in patients with non-small cell lung cancer (NSCLC), 108 NSCLC patients, including 40 patients with benign lung diseases and 30 healthy controls, were investigated. The serum VEGF levels were detected by ELISA and CK19 mRNA in peripheral blood by reverse transcriptase-polymerase chain reaction (RT-PCR). In NSCLC group, the serum VEGF levels and the positive rate of CK19 mRNA in peripheral blood were 479.8±268.5 pg/mL and 66.7%, which were significantly higher than those of the other two groups respectively (P<0.01), and both of them were increased significantly with the progression of clinical stage of the tumors (P<0.01). Serum VEGF levels as well as the positive rate of CK19 mRNA in different pathological types of lung cancer had no significant differences (P>0.05). Serum VEGF levels in the patients positive for CK19 mRNA was 561.7±325.6 pg/mL. It is significantly higher than that in the negative patients (P<0.01). There existed a significant correlation between serum VEGF levels and expression of CK19 mRNA in peripheral blood in NSCLC patients (P<0.001). The detection of serum VEGF levels and CK19 mRNA in peripheral blood is helpful in judging the condition and the prognosis of NSCLC patients, and serum VEGF levels and CK19 mRNA are independent of the pathological types of lung cancer. The micrometastasis in peripheral blood of NSCLC patients is significantly associated with serum VEGF levels.

This study explored the role of apoptosis of alveolar wall cells of chronic obstructive pulmonary disease (COPD) patients with pulmonary emphysema in the pathogenesis of emphysema. The subjects were divided into three groups: COPD patients with pulmonary emphysema (COPD group), asymptomatic smokers and non-smokers. Lung tissues were harvested and histologically assessed. TUNEL assay was employed to determine the apoptotic cells. The expression of PCNA, Bax and SP-C in the lung alveolar wall cells were immunohistochemically determined. SP-C immunofluorescence staining was used to identify type II alveolar cells in the TUNEL-positive cells. The mean linear interval (MLI), mean alveoli number (MAN) and mean alveoli area (MAA) in COPD group were significantly different as compared with those in asymptomatic smokers and non-smokers, respectively (P<0.01). The proliferation index (PI), apoptosis index (AI) and the percentage of Bax-positive cells in COPD group were significantly greater than those of asymptomatic smokers and non-smokers (P<0.01). However, the percentage of SP-C-positive cells was significantly lower in COPD group than in asymptomatic smokers and non-smokers (P<0.01). Most of the TUNEL-positive cells expressed SP-C. In COPD group, the apoptosis of alveolar wall cells, especially apoptosis of type-II cells, may take part in the pathogenesis of emphysema. Up-regulation of Bax expression may be responsible for the apoptosis of alveolar wall cells in the COPD patients with pulmonary emphysema.

To establish a stable and reliable model of refractory hypoxemia acute respiratory distress syndrome (ARDS) and examine its pathological mechanisms, a total of 144 healthy male Wistar rats were randomized into 4 groups: group I (saline control group), group II (LPS intravenous “single-hit” group), group III (LPS intratracheal “single-hit” group) and Group IV (LPS “two-hit” group). Rats were intravenously injected or intratracheally instilled with a large dose of LPS (10 mg/kg in 0.5 mL) to simulate a single attack of ARDS, or intraperitoneally injected with a small dose of LPS (1 mg/kg) followed by tracheal instillation with median dose of LPS (5 mg/kg) to establish a “two-hit” model. Rats in each group were monitored by arterial blood gas analysis and visual inspection for three consecutive days. Arterial blood gas values, lung wet/dry weight ratio and pathological pulmonary changes were analyzed to determine the effects of each ALI/ARDS model. Concentrations of TNF-α, IL-1 and IL-10 in the bronchoalveolar lavage fluid (BALF) and blood plasma were measured by using enzyme-linked immunosorbent assays (ELISA). Our resulsts showed that single LPS-stimulation, whether through intravenous injection or tracheal instillation, could only induce ALI and temporary hypoxemia in rats. A two-hit LPS stimulation induces prolonged hypoxemia and specific pulmonary injury in rats, and is therefore a more ideal approximation of ARDS in the animal model. The pathogenesis of LPS two-hit-induced ARDS is associated with an uncontrolled systemic inflammatory response and inflammatory injury. It is concluded that the rat ARDS model produced by our LPS two-hit method is more stable and reliable than previous models, and closer to the diagnostic criteria of ARDS, and better mimics the pathological process of ARDS.

To investigate the molecular mechanism by which Tanshinone IIA (TSN IIA) prevents left ventricular hypertrophy (LVH), we examined the expression of AT1R, TGF-β1 and Smads gene in the hypertrophic myocardium of hypertensive rats with abdominal aorta constriction. LVH model was established by creating abdominal aorta constriction. Four weeks later, animals were randomly divided into 4 groups with 8 animals in each. One group was used as model control, the other three groups were treated with TSN IIA (20 mg/kg), TSN IIA (10 mg/kg) and valsartan (10 mg/kg), respectively. Another 8 SD rats were subjected to sham surgery and served as blank control. After 8-week treatment, the caudal artery pressure of the animals was measured. The tissues of left ventricle were taken for the measurement of the left ventricular mass index (LVMI) and pathological sectioning and HE-staining were used for determining the myocardial fiber dimension (MFD). The mRNA expression of AT1R, protein expression of TGF-beta1 and activity of Smad-2, 4, 7 were detected by RT-PCR and Western blotting, respectively. Our results showed that (1) the blood pressure of rats treated with TSN IIA, either at high or low dose, was significantly higher than those in the control and valsartan-treated group (P<0.01, P<0.05); (2) LVMI and MFD in TSN IIA and valsartan-treated rats were higher than those in the control group (P<0.05) but significantly lower than those in the model control (P<0.01); (3) the high doses of TSN IIA and valsartan significantly down-regulated the mRNA expression of AT1R and protein expression of TGF-beta1 and Smad-3 in the hypertrophic myocardium (P<0.01), and TGF-beta1 in valsartan-treated animals was more significantly lower than that in rats treated with TSN IIA; (4) the two doses of TSN IIA and valsartan significantly up-regulated the protein expression of Smad-7 in the hypertrophic myocardium (P<0.01), and Smad-7 in the animals treated with high-dose TSN IIA was significantly higher than that in rats treated with valsartan. It is concluded that inhibition of myocardial hypertrophy induced by TSN IIA independent of blood pressure. The underlying mechanism might be the down-regulated expression of AT1R mRNA and Smad-3, increased production of Smad-7, and blocking effect of TSN IIA on TGF beta1/Smads signal pathway in local myocardium.

Trichinella spiralis infection in rodents is a well-known model of intestinal inflammation associated with hypermotility. The aim of the study was to use this experimental model to elucidate if Th17 cells are involved in the development of gastrointestinal hypermotility. Colonic smooth muscle contractility was investigated in response to acetylcholine. The levels of IL-17, IL-23 and TGF-β1 in colon were measured by Western blotting. Flow cytometric detection of intracellular IFN-γ/IL-4/IL-17 cytokine production was used to analyze the proportions of CD4+ T cells subsets in colon. Our results showed that colonic muscle contractility was increased 2 weeks post infection (PI) and stayed high 12 weeks PI when no discernible inflammation was present in the gut. The proportion of Th17 cells and the expression of IL-17 were up-regulated in colon 2 weeks PI and returned to normal 8 weeks PI. The content of IL-17 was correlated with the colonic smooth muscle hypercontracility 2 weeks PI. Meanwhile, TGF-β1 was increased 2 weeks PI, while IL-23 was normal. Our results suggest that Th17 cells affect the colonic muscle contractility in mice infected with Trichinella spiralis at intestine stage but not at muscle stage and the effect of Th17 cells on muscle contractility might be induced by TGF-β1. Other cytokines might be involved in the hypercontracility of colonic smooth muscle at muscle stage.

The purpose of this study was to investigate bacterial translocation and change in intestinal permeability in patients after abdominal surgery. Sixty-three patients undergoing elective abdominal surgery were enrolled in the study. Blood samples were collected prior to operation and 2, 24, 48 h after surgery for bacterial culture, microbial DNA extraction, plasma D-lactate and endotoxin measurement. PCR analysis was performed after DNA extraction, with β-lactosidase gene of E. coli and 16S rRNA gene as target genes. All patients were observed for a period of 30 days for infectious complications. Our results showed that no bacterial DNA was detected before surgery, but after operation it was found in 12 patients (19.0%). Bacterial DNA was detected in 41.7% (10/24) of SIRS patients and 5.1% (2/39) of non-SIRS patients (P<0.01). About 83.3% of PCR-positive patients developed systemic inflammatory response syndrome (SIRS), but only 27.5% of PCR-negative patients did so (P<0.01). Two thirds of PCR-positive patients developed infectious complications, while none of PCR-negative patients did (P<0.01). The blood culture was positive only in 3 patients (4.8%), who were all PCR-positive. E. coli DNA was found in 66.7% of the PCR-positive patients. The plasma levels of D-lactate and endotoxin were elevated significantly 2, 24 and 48 h after operation in PCR-positive patients, with a significant positive correlation found between them (r=0.91, P<0.01). It is concluded that increased intestinal permeability was closely related with bacterial translocation. Intestinal bacterial translocation (most commonly E. coli) might occur at early stage (2 h) after abdominal surgery. Postoperative SIRS and infection might bear a close relationship with bacterial translocation.

To observe the chelation of GRP78 with lead (Pb) and its localization changes, astroglial cells from Wistar rat brain were primarily cultured in medium with acetate Pb. The processes were terminated at different time points. The immunoprecipitation (IP) and Western blotting were used for GRP78 purification and expression and the Pb concentration was determined by employing atomic absorption spectrophotometry (AAS). The localization change of GRP78 was observed with colloid gold immunoelectron microscopy. The results showed that the expression of GRP78 was increased significantly in the cells treated with 1.0 μmol/L acetate Pb for 24 h and peaked at 96–192 h (P<0.01), and at the 12th day, the expression of GRP78 began to decrease but was still higher than normal (P<0.05). Pb content started to increase when cells were treated by acetate Pb for 24 h, and the peak appeared at 8 day (P<0.01), and then Pb content decreased gradually, but was still higher than normal (P<0.05). GRP78 protein expression began to remarkably increase when it transferred from ER to the cytosol around the nuclei 24 h after treatment with Pb. It is concluded that GRP78 in astroglia could strongly chelate with Pb ions and it might be a target protein of Pb.

Hypermethylation in the promoter region of tumor suppressor genes is a common mechanism of gene silencing, which tends to occur in cancer. The effects of 5-Aza-2′-deoxycytidine (5-Aza-CdR), a specific DNA methyltransferase inhibitor, on the cell proliferation of human breast cancer cell line MCF-7 and on the expression of Apaf-1 gene were investigated. Human MCF-7 cells were incubated with increasing concentrations of 5-Aza-CdR for 12 to 120 h. The growth inhibition rates of MCF-7 cells were detected by MTT assay. Changes of cell cycle distribution and apoptotic rates of MCF-7 cells were determined by flow cytometry. The expressions of DNA methyltransferase 3b mRNA and Apaf-1 mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, the expression of Apaf-1 protein was detected by Western blotting. The results showed that 5-Aza-CdR significantly inhibited the growth of MCF-7 cells and the growth inhibition rate of MCF-7 cells was significantly enhanced with the concentration of 5-Aza-CdR and the action time. Flow cytometry indicated that 5-Aza-CdR could significantly induce G₁/S cell cycle arrest and increase the apoptosis rate of MCF-7 cells. The mRNA and protein expressions of Apaf-1 were up-regulated in MCF-7 cells treated with 5-Aza-CdR, which was accompanied by down-regulation of DNA methyltransferase 3b mRNA. It is concluded that 5-Aza-CdR might retard the growth of tumor cells and promote the apoptosis of MCF-7 breast cancer cells by inhibiting the expression of DNA methyltransferase 3b and re-activating the Apaf-1 gene expression.

The cell adhesive properties of decellularized valve scaffolds were promoted by immobilization of valve scaffold with arginine-glycine-aspartic acid (RGD)-containing peptides. Porcine aortic valves were decellularized with trypsin/EDTA, and detergent Triton X-100. With the help of a coupling reagent Sulfo-LC-SPDP, the valve scaffolds were immobilized with glycine-arginine-glycine-aspartic acid-serine-proline-cysteine (GRGDSPC) peptide. X-ray photoelectron spectroscopy (XPS) was used for surface structure analysis. Myofibroblasts harvested from rats were seeded onto the valve scaffolds. Cell count by using microscopy and modified MTT assay were performed to assess cell adhesion. Based on the spectra of XPS, the conjugation of GRGDSPC peptide with decellularized valve scaffolds was confirmed. Both cell count and MTT assay showed that myofibroblasts were much easier to adhere to the modified valve scaffolds, which was also confirmed histologically. Our findings suggest that it is feasible to immobilize RGD-containing peptides onto decellularized valve scaffolds. And the technique can effectively promote cell adhesion, which is beneficial for in vitro tissue engineering of heart valves.

In this study, a novel polyurethane membrane, modified by superfine silk-fibroin powder, was prepared for small-diameter vascular grafting. Scanning electron microscopy, transmission electron microscopy, and histological examination were applied to evaluate histocompatibility of this polyurethane membrane. The polyurethane membrane was compared with polytetrafluoroethylene material. A pseudomembrane and gap formed between polytetrafluoroethylene and the surrounding tissues, and no cells infiltrated or grew into the polytetrafluoroethylene material. On the contrary, superfine silk-fibroin powder/polyurethane blend membrane merged tightly with the surrounding tissues without gaps, and cells infiltrated and grew into the material. Moreover, the negative effects of superfine silk-fibroin powder/polyurethane blend membrane on cells were less than those of its polytetrafluoroethylene counterpart. Our findings indicated that the superfine silk-fibroin powder/polyurethane blend membrane has better histocompatibility than polytetrafluoroethylene membrane. It is concluded that the superfine silk-fibroin powder/polyurethane blend membrane is a promising biomaterial for small-diameter prosthesis.

To synthesize KLD-12 peptide with sequence of AcN-KLDLKLDLKLDL-CNH₂ and trigger its self-assembly in vitro, to encapsulate rabbit MSCs within peptide hydrogel for 3-D culture and to evaluate the feasibility of using it as injectable scaffold for tissue engineering of IVD. KLD-12 peptide was purified and tested with high performance liquid chromatography (HPLC) and mass spectroscopy (MS). KLD-12 peptide solutions with concentrations of 5 g/L, 2.5 g/L and 1 g/L were triggered to self-assembly with 1×PBS in vitro, and the self-assembled peptide hydrogel was morphologically observed. Atomic force microscope (AFM) was employed to examine the inner structure of self-assembled peptide hydrogel. Mesenchymal stem cells (MSCs) were encapsulated within peptide hydrogel for 3-D culture for 2 weeks. Calcein-AM/PI fluorescence staining was used to detect living and dead cells. Cell viability was observed to evaluate the bioactivity of MSCs in KLD-12 peptide hydrogel. The results of HPLC and MS showed that the relative molecular mass of KLD-12 peptide was 1467.83, with a purity quotient of 95.36%. KLD-12 peptide at 5 g/L could self-assemble to produce a hydrogel, which was structurally integral and homogeneous and was able to provide sufficient cohesion to retain the shape of hydrogel. AFM demonstrated that the self-assembly of KLD-12 peptide hydrogel was successful and the assembled material was composed of a kind of nano-fiber with a diameter of 30–40 nm and a length of hundreds of nm. Calcein-AM/PI fluorescence staining revealed that MSCs in KLD-12 peptide hydrogel grew well. Cell activity detection exhibited that the A value increased over the culture time. It is concluded that KLD-12 peptide was synthesized successfully and was able to self-assemble to produce nano-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.

To explore a new way of constructing bioartificial renal tubule assist device (RAD) in vitro and its function of transporting sodium (Na⁺) and glucose and to evaluate the application of atomic force microscope in the RAD construction, rat renal tubular epithelial cell line NRK-52E was cultured in vitro, seeded onto the outer surfaces of hollow fibers in a bioreactor, and then cultured for two weeks to construct RAD. Bioreactor hollow fibers without NRK-52E cells were used as control. The morphologies of attached cells were observed with scanning electron microscope, and the junctions of cells and polysulfone membrane were observed with atomic force microscope. Transportation of Na⁺ and glucose was measured. Oubaine and phlorizin were used to inhibit the transporting property. The results showed that NRK-52E cells and polysulfone membrane were closely linked, as observed under atomic force microscope. After exposure to oubaine and phlorizin, transporting rates of Na⁺ and glucose were decreased significantly in the RAD group as compared with that in the control group (P<0.01). Furthermore, when the inhibitors were removed, transportation of Na⁺ and glucose was restored. It is concluded that a new RAD was constructed successfully in vitro, and it is able to selectively transport Na⁺ and glucose.

To analyze MR manifestations of postoperative spinal complications and investigate the value of MRI in the diagnosis and differentiated diagnosis, 114 cases of spinal postoperative complications were analyzed retrospectively and compared with the clinical data. The results showed that the main postoperative spinal complications included spinal stenosis (n=33, consisting of 21 cases of epidural fibrosis and 12 cases of epidural hematoma or epidural abscess), lack of spinal stability (n=43), infection (n=23, consisting of 7 cases of para-spinal soft-tissue infection, 5 cases of vertebral discitis, 4 cases of vertebral and appendix infection, 3 cases of epidural abscess, 2 cases of myelitis, 2 cases of spinal arachnoiditis), others (n=28, consisting of 12 cases of inner fixation failure, 9 cases of epidural hematoma, 7 cases of cerebrospinal fluid gusher). It is concluded that MRI can specifically display all kinds of postoperative spinal complications, and is of significant value in the diagnosis and differentiated diagnosis of postoperative spinal complications.