To clarify the role of TAFI in hypertensive disorders in pregnancy, 22 subjects, including 10 with pre-eclampsia (PE) and 12 with gestational hypertension were examined for the levels of TAFI and thrombin-antithrombin (TAT) complex. Thirty normal pregnant women served as controls. ELISA was employed for the detection. The results showed that the TAFI antigen levels in normal pregnancy group, gestational hypertension group and PE group were (85.35±24.69)%, (99.65±18.27)%, (110.12±23.36)%; (97.06±21.40)%, (114.08±27.76)%, (125.49±24.70)%; (106.6±19.21)%, (129.2±25.07)%, (139.1±30.12)%, in the 1st, 2nd and 3rd trimester respectively. No significant differences were found between the normal pregnancy group and gestational hypertension group but significant difference existed between normal pregnancy group and PE group in each trimester (P<0.05). TAT complexes were significantly higher in patients with PE than that in controls (P<0.05), but no correlation was found between TAT and TAFI. It is concluded that TAFI may contributed to the impairment of fibrinolysis in the patients with PE and may serves as a sensitive indicator for PE, but it may not help in the diagnosis of the gestational hypertension.
Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke extract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type II and its relationship with P21WAF1, the alveolar epithelial type II cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type II cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke extract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose-and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic microscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1 phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type II and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke extract-induced inhibition of cell proliferation.
To investigate the disposition and tissue distribution of ML12 after intravenous (iv) administration in rats, the compound in plasma or in tissue was extracted into ethyl acetate under basic condition and was determined by HPLC after extracted by dilute sulfuric acid. Excitation wavelength and emission wavelength of fluorescence detection were 278 nm and 307 nm, respectively. The data were processed with the software 3P97 to calculate the main pharmaceutical parameters of ML12. At dose of 5 and 10 mg/kg, the elimination of the drug from plasma was found to be kinetically linear, but when the dosage was 20 mg/kg, a non-linear feature was observed. The highest level of ML12 was found in the kidney. Distribution of ML12 after iv administration was extensive and the concentration-time profile was found to be fitted to an open two-compartment model.
To study the effect of titanium alloy cage on the treatment of the ischemic necrosis of femoral head in dog, the model of the ischemic necrosis of femoral head was made with the liquid nitrogen in 15 hybrid adult dogs. The titanium alloy cage made of a hollow cylinder was driven into the subchondral bone of necrotic femoral head via central channel. The dogs were divided into 3 groups, each group was sacrificed 3, 6, 12 weeks after the operation respectively. No collapse of femoral head was observed after the operation. The position of the cages was good on radiograph. Microscopically, the cancellous bone of necrotic femoral head rebuilt gradually and grew into cage. After 12 weeks of creeping substitution, the cancellous bone filled up the hollow cavity and holes of the cages. It is concluded that the titanium alloy cage can provide structural support for the subchondral bone and prevent collapse and can be used for the treatment of the ischemic necrosis of femoral head.
To study the apoptosis induced by cisplatin in cervical cancer cell line HeLa and its mechanism, cell growth inhibition of cisplatin on HeLa cells was analyzed by MTT assay. Cell apoptosis was examined by cytometry and Hoechst33258 staining after treatment with cisplatin. The effects of cisplatin on transcription of E6 were analyzed by RT-PCR. The protein expressions of E6, p53, p21, Bax and Bcl-2 were studied by Western blotting. Cisplatin inhibited proliferation in a time-and dose-dependant manner. Cytometically, sub-G1 peak showed higher apoptosis rates in the experimental group than those in the control. Hoechst33258staining exhibited apoptosis induced by cisplatin. RT-PCR revealed that cisplatin decreased transcription of E6. Western blotting showed that cisplatin decreased protein expression of E6 and increased protein expression of p53, p21 and Bax. It had no effect on protein expression of Bcl-2. It is concluded that cisplatin can induce apoptosis in HeLa cells by suppressing HPV E6 and thereby restoring the function of p53.
To investigate the effects of hyperoxia on mitochondrial multienzyme complex III (cytochrome, Cytb) and V (ATPase6, 8) in premature newborn rat lung, the 1-day-old preterm SD rats were randomly assigned to hyperoxia group and air group. The rats in hyperoxia group were continuously exposed to 85% oxygen and those in air group to room air. After 1, 4, 7, 10, 14 day(s) of exposure, these rats were killed, total lung RNA was extracted and Cytb, ATPase6, 8 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR). Western blotting was used to detect the expression of Cytb protein in lung tissue. The results showed that compared with air group, Cytb mRNA expression was significantly increased (P>0.05) after 1, 4 day(s) of exposure. The general tendency decreased after 7 days, and its expression became weak but difference in mRNA expression between the two groups was not significant (P>0.05). ATPase6 mRNA expression was significantly increased 1 day after the exposure (P<0.05) and did not show any significant change 4, 7, 10 days after the exposure (P>0.05). At the 14th day, ATPase6 mRNA expression was significantly increased (P<0.05). ATPase8 mRNA expression did not show any significant change 1, 4, 10 day(s) after the exposure (P>0.05). At the 7th and 14th day, ATPase8 mRNA expression was significantly increased (P<0.05). Western blotting showed that Cytb protein expression was increased 1,4 day(s) after the exposure, but the difference between the two groups was not significant (P>0.05). The general tendency was decreased after 7 days, and its expression became weak but difference was not significant 7, 10 days after the exposure (P>0.05). At day 14 its expression became significantly weak (P<0.05). We are led to conclude that exposure to high concentrations of oxygen can significantly change the expression of Cytb and ATPase6, 8, which results in uncoupling of oxidative phosphorylation in mitochondrial respiration chain, and plays an important role in the mechanism of hyperoxia-induced lung injury.
Three plasmids (pGenesil-P1, pGenesil-P2, pGenesil-P3) with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells (bCEC) were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control (pGenesil-HK). The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group (non-transfected group). pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry (FCM). Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference (RNAi) may be an effective means to promote the proliferation of CEC.
Mouse blastocysts were exposed to doses of 0, 1 and 10 μmol/L retinoic acid (RA) for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL-FITC) assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm (TE) and inner cell mass (ICM) cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA (10 μmol/L) resulted in a more significant apoptosis and higher expression level of Fas proteins (P<0.01). It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.
To investigate whether glutamate and voltage-gated calcium channels-independent calcium influx exists during acute anoxic neuronal damage and its possible relationship to neuronal protective function of NGF. In in vitro model of acute anoxia, hippocampal cultures from newborn rats were exposed to 3 mmol/L KCN. Changes of intracellular Ca2+ concentration ([Ca2+]i) were monitored by con-focal imaging and cell viability was assayed by PI and cFDA staining. The results showed that after treatment with primary hippocampal cultures with 3 mmol/L KCN for 15 min, [Ca2+]i was significantly increased 6.27-fold compared to pre-anoxia level and 73.3% of the cells died. When combination of 20 μmol/L MK-801 (glutamate receptor antagonist), 40 μmol/L CNQX (AMPA receptor antagonist) and 5 μmol/L nimodipine (voltage-gated calcium channel antagonist) (hereafter denoted as MCN) were administrated to hippocampal cultures, levels of [Ca2+]i and cell death rate induced by KCN were partially reduced by 35.9% and 47.5% respectively. However, Gd3+ (10 μmol/L) almost completely blocked KCN-mediated [Ca2+]i elevation by 81.9% and reduced neuronal death by 88.8% in the presence of MCN. It is noteworthy that NGF, used in combination with MCN, inhibited KCN-induced [Ca2+]i increase by 77.4% and reduced cell death by 87.1%. Only PLC inhibitor U73122 (10 μmol/L) abolished NGF effects. It is concluded that Gd3+-sensitive calcium influx, which is NMDA (glutamate receptor) and voltage-gated calcium channels-independent, is responsible for acute anoxic neuronal death. NGF can inhibit Gd3+-sensitive calcium influx and reduce anoxic neuronal death through activating PLC pathway.
In order to investigate the effects of aconitine on [Ca2+] oscillation patterns in cultured myocytes of neonatal rats, fluorescent Ca2+ indicator Fluo-4 NW and laser scanning confocal microscope (LSCM) were used to detect the real-time changes of [Ca2+] oscillation patterns in the cultured myocytes before and after aconitine (1.0 μmol/L) incubation or antiarrhythmic peptide (AAP) and aconitine co-incubation. The results showed under control conditions, [Ca2+] oscillations were irregular but relatively stable, occasionally accompanied by small calcium sparks. After incubation of the cultures with aconitine, high frequency [Ca2+] oscillations emerged in both nuclear and cytoplasmic regions, whereas typical calcium sparks disappeared and the average [Ca2+] in the cytoplasm of the cardiomyocyte did not change significantly. In AAP-treated cultures, intracellular [Ca2+] oscillation also changed, with periodic frequency, increased amplitudes and prolonged duration of calcium sparks. These patterns were not altered significantly by subsequent aconitine incubation. The basal value of [Ca2+] in nuclear region was higher than that in the cytoplasmic region. In the presence or absence of drugs, the [Ca2+] oscillated synchronously in both the nuclear and cytoplasmic regions of the same cardiomyocyte. It was concluded that although oscillating strenuously at high frequency, the average [Ca2+] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incubation, compared to the controls. The observations indicate that aconitine induces the changes in [Ca2+] oscillation frequency other than the Ca2+ overload.
Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegenerative diseases, we packed recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.