Objective To investigate the effects of hypertonic saline (HTS) treatment on the function and susceptibility to sepsis of reticuloendothelial system (RES) in mice with hemorrhagic shock. Methods Forty percent of total blood volume of male Balb/c mice was withdrawn by cardiac puncture. Two hours later, the mice were treated with blood infusion and normal saline (10 ml/kg) or 7.5% NaCl (10 ml/kg).The survival rate of the mice was observed after cecal ligation and puncture (CLP). The phagocytosis function of the RES was measured by carbon clearance rate(α) and carbon amount ingested by the macrophages of liver and spleen. In vitro, the peritoneal phagocyte function in solutions of different osmotic pressor was measured by assaying neutral red amount taken in. Results The survival rate after CLP in HTS treated group was 70%, whereas all the mice in the normal saline group died. At the third hour after hemorrhagic shock, the RES carbon clearance rate(α) and carbon amount ingested by the macrophages of liver in the HTS treated mice were 5.61±0.42 and 0.59±0.19 respectively, significantly higher than those in the normal saline treated mice (4.15±0.62， 0.42±0.16). In vitro, hyperosmolarity below 40 mmol/L had no significant effects on the phagocytosis activity of peritoneal macrophages in mice. Conclusion Treating hemorrhagic shock with HTS can decrease the susceptibility to sepsis and improve the RES phagocytosis function indirectly.
ObjectiveTo establish a mouse model of acute necrotizing pancreatitis.MethodsThirty-six male ICR mice were randomly divided into control group (n=6) and experimental group (n=30). Each of the animals in the experimental group received 7 intraperitoneal injections of caerulein (50 μg/kg body weight) in 0.9% NaCl at hourly intervals over 6 hours. The animals in the experimental group were killed at 9,18,24,48 and 72 hours respectively after the first caerulein injection. The control animals received the same volume of 0.9% NaCl without caerulein. The animals in the control group were killed at the 18th hour after the first intraperitoneal injection. The severity of acute necrotizing pancreatitis was evaluated in terms of amylase level, pancreatic weight/body weight and the histological changes. Variance analysis was employed in the processing of these data. ResultsBoth amylase level and pancreatic weight elevated 9 hours after the first caerulein injection, and correlated with the course of pancreatitis. The maximums of both alterations were observed at the same time point (18 hours after the first injection of caerulein). Prominent interstitial inflammation and acinar cell necrosis occurred at the 18th hour, and the histological score for pancreatitis reached a maximum （P＜0.05）. Conclusion Intraperitoneal injection of a large dosage of caerulein can induce acute necrotizing pancreatitis in ICR mice. This method is simple and noninvasive, and the model established thus is stable and reproducible.
Objective To investigate the gene expression of beta-defensin-4 (mBD-4) and mBD-6 in acute lung injury (ALI) mouse.Methods Sixty adult mice were randomly divided into a control group and a ALI group.ALI was induced by intraperitoneal injection of lipopolysaccharide (LPS) in the ALI group.The control group was treated with same dose of normal saline.The lung tissues were harvested at different time point after stimulation.The expression of mBD-4 and mBD-6 mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction.DNA sequencing was used to confirm the specificity of mBD-4 and mBD-6 cDNA fragment.Results There were no obvious mBD-4 and mBD-6 mRNA expression in mouse lung in the control group at all time points and ALI 6 h group.In the ALI group a marked increasing expression was found on 12 h，1 d and 3 d after LPS stimulation.The mBD-4 mRNA expression was significant higher in the ALI groups of 1 d and 3 d points than that of ALI 12 h group with no obvious difference between each other.There were no significant differences of mBD-6 mRNA expression between ALI groups of 12 h，1 d and 3 d points Conclusion mBD-4 and mBD-6 mRNA is not constitutive expressed in mouse lung and show a up-regulative expression pattern after ALI.
Objective To observe the expression of adenovirus vector coding for mouse endostatin gene（Ad-mES） in lung cancer cells and its antiangiogenic activity in human umbilical vein endothelial cells（ECV304） in vitro.Methods Lewis lung cancer（LLC） cells were transfected with Ad-mES at different multiplicity of infection（MOI）.The expression of mES in LLC cells and supernatant after 48 hours was detected by immunohistochemical staining and Western blot respectively.The inhibitory effect of supernatant at different MOI on ECV304 non-stamulated and stimulated by basic fibroblast growth factor（bFGF） was measured by methyl thiazolyl tetrazolium（MTT） assay.Results After transfected for 48 hours，endostatin was identified in the cell plasma of infected LLC and negative result was founded in non-infected LLC.Western blot revealed band of endostatin in 20 kDa in culture supernatant of infected LLC and negative results in non-infected LLC.The inhibitory effects on ECV304 cell proliferation were ber at higher MOI，and the difference was significant between stimulated and non-stamulated cells by bFGF（Plt;0.05）.Conclusion Ad-mES can transfect and express endostatin effectively in LLC with biological activity
Objective To investigate the serum levels of endostatin and vascular endothelial growth factor ( VEGF) at different therapy stages of mouse Lewis lung carcinoma, and elucidate the relation to the progress and prognosis of tumor. Methods Forty-four Lewis lung carcinoma-bearing C57BL/6 mice were randomly divided into 4 groups, ie. a non-therapy group, a chemotherapy group, a gene therapy group, and a combination therapy group ( chemotherapy plus gene therapy) . Eleven healthy mice were included as normal control group. Serum was collected on the 0th, 5th, 19th day after therapy for measurement of endostatin and VEGF by ELISA. The correlations were analysed between endostatin and VEGF levels in each group. Results ( 1) The serum endostatin levels had no significant difference in all groups on the 0th day,but increased significantly on the 5th day in the gene and combination therapy groups than those in other three groups ( all P lt;0. 01) . Then the endostatin level decreased on the 19th day in the gene and combination therapy groups, but still higher than those in the chemotherapy group and the normal group. ( 2 ) On the contrary, serum VEGF levels of the gene and combination therapy groups decreased significantly on the 5th day and increased little on the 19th day, which were both significant lower than those in chemotherapy group on the 5th and 19th day( all P lt; 0. 05) . There were significant diferences between the three therapy groups and the non-therapy group( all P lt;0. 05) . ( 3) Negative correlations between VEGF and endostatin levels were revealed in the gene and combination therapy groups ( r = - 0. 77 and - 0. 761 respectively) .Correlation was not found in the non-therapy and chemotherapy groups. Conclusion The serum levels of endostatin and VEGF might be used as monitoring indices of antiangiogenesis therapy.
Objective To investigate the expressions of β1, 3-N-acetyl glucosaminyl transfrases ( Fringe) ( RFNG, LFNG and MFNG) in lung tissues and lung T cells isolated from asthmatic mice, and to explore the role of Fringe in pathogenesis of asthma. Methods Asthmatic BALB/ c mouse model was established by inhalation of ovalbumin after intraperitoneal injection. Meanwhile, the control groups were established by normal saline. Lung tissues were sampled after 24 hours since the last stimulation. T cells were isolated from the lung tissues using percol and NylonFiber. The mRNA expressions of three kinds of Fringe in the lung tissues and lung T cells were examined by reverse transcription-PCR ( RT-PCR) . The protein expressions of Fringe in the lung tissues were detected by Western blot. Results The mRNA expressions of RFNG, LFNG and MFNG were detectable in the lung tissues and lung T cells. The mRNA expressions of RFNG increased in the asthmatic group compared with the control group( lung tissues: 0. 92 ±0. 35 vs 0. 51 ±0. 13, P lt; 0. 01; lung T cells: 0. 33 ±0. 06 vs 0. 18 ±0. 07, P lt; 0. 01) . LFNG mRNA had lower expression level in the asthmatic group( lung tissue: 0. 77 ±0. 32 vs 1. 61 ±0. 31, P lt; 0. 01; lung T cells: 0. 49 ±0. 19 vs 0. 71 ±0. 03, P lt;0. 01) . No difference on the mRNA expression of MFNG was found in the lung tissues( 1. 44 ±0. 29 vs 1. 70 ±0. 44, P gt; 0. 05) . MFNG mRNA expression decreased in the asthmatic group compared with the control group in the T cells( 1. 17 ±0. 04 vs 0. 68 ±0. 07, P lt;0. 05) . The results of western blot were consistent with RT-PCR results of the lung tissues. The expressions of RFNG increased in the asthmatic group( 1. 17 ±0. 04 vs 0. 68 ±0. 07, P lt;0. 05) . The expression of MFNG has no difference between two groups( 8. 10 ±0. 60 vs 9. 12 ±0. 07, P gt;0. 05) . LFNG had a lower expression in the asthmatic group( 4. 11 ±0. 38 vs 6. 41 ±0. 11, P lt; 0. 05) . Conclusion The abnormal expressions of three kinds of Fringe may be involved in the pathogenesis of asthma.
Objective To establish a beta 2 adrenergic receptor ( β2 R) down-regulative asthmatic model, to explore the mechanism of β2 R down-regulation and effectiveness of corticosteroids. Methods Thirty-two BALB/c mice were divided into four groups, ie. a control group, an asthmatic group, a β2R downregulative group, and a dexamethasone group. The asthmatic group, the β2 R down-regulative group and the dexamethasone group were sensitized on 0th, 14th and 21th day by intraperitoneal injection of ovalbumin ( OVA) together with aluminumhydroxide in a total volume of 200 μL. Fromthe 28th day on, the mice were challenged with an aerosol of 1% OVA( W/V) in saline using an ultrasonic nebulizer 30 min/d for a week.The β2 R down-regulative group and the dexamethasone group underwent the same procedure as the asthmatic group besides daily intraperitoneal injection of 60 μg salbutamol and inhaling an aerosol of 0. 01%salbutamol 30 min/d for a week half an hour before challenged with OVA. The dexamethasone group was injected dexamethasone 5 mg·kg- 1·d - 1 for a week by intraperitoneal injection on the basis of OVA challenge and salbutamol intervention. The control group was sensitized and challenged with PBS. Airway resistance was measured by plethysmography. IL-4 and IFN-γlevels in BALF, and total IgE concentration in serum were measured by ELISA. Total and differential cell counts in bronchial alveolar lavage fluid ( BALF)were measured. Total amount and number of β2 R in lung tissue were evaluated by immune blotting analysis and radioligand receptor binding assay, respectively. Results Compared to the control group and the dexamethasone group, airway resistance of the asthmatic group and the β2 R down-regulative group increasedobviously provocated by a high dose of acetylcholine ( P lt;0. 01) . Eosinophil, neutrophil, lymphocyte counts in BALF, IL-4 level in BALF, and total IgE in serumincreased significantly also ( P lt;0. 01) , while IFN-γin BALF decreased significantly. Compared to the control group, the asthmatic group and the dexamethasonegroup, the total amount and number of β2 R significantly decreased in the β2 R down-regulative group ( P lt;0. 01) , while no significant difference was found among the control group, the asthmatic group and the dexamethasone group. Conclusions β2 R down-regulative asthmatic model can be successfully establishedby peritoneal injection and inhalation of salbutamol on the basis of OVA sensitization and challenge.Dexamethasone can prevent the down-regulation of β2 R.
Objective To establish a mouse model of acute lung injury ( ALI) and pulmonary fibrosis by low dose lipopolysaccharide ( LPS) intermittent intraperitoneal injection, and to explore the pathogenesis of ALI and pulmonary fibrosis induced by endotoxin. Methods Forty C57BL/6 mice were randomly divided into a control group, a 3-days LPS group, a 2-weeks LPS group, and a 4-weeks LPS group,with 10 mice in each group. LPS was injected intraperitoneally at dose of 5 mg/ kg for three consecutive daysin the three LPS groups. Equivalent normal saline was injected by the same way in the control group. The mice lung tissues were obtained respectively 3 days ( the control group and 3-days LPS group) , 2 weeks ( the 2-weeks LPS group) , and 4 weeks ( the 4-weeks LPS group) after LPS or saline stimulation. HE staining,Van-Gieson collagen staining, and Ashcroft fibrosis score assessment were applied to evaluate the development of inflammation and fibrosis in lung tissue at various stages of ALI after LPS-stimulation. The mRNA expression of type Ⅰ procollagen and alpha smooth muscle actin ( α-SMA) were detected by realtime PCR. The deposition of collagen and fibrosis in lung tissue were detected by hydroxyproline assay. The survival condition of each group was also recorded. Results Acute inflammation occurred in mice lung tissue 3 days after intraperitoneal injection of LPS. Collagen deposited in pulmonary interstitium2 weeks afterLPS-stimulation and formed typical pulmonary interstitial fibrosis 4 weeks later accompanying with increase of Ashcroft fibrosis score. Real-time PCR and hydroxyproline assay showed that the expression of collagen and α-SMA increased 3 days after LPS-stimulation and reached the peak 4 weeks later. The animals were all survived up to the endpoint of experiment. Conclusions Accompanying with inflammation, pulmonary fibrosis initiated at early stage of ALI induced by LPS. Intraperitoneal injection of LPS at dose of 5 mg/kg for three consecutive days was able to establish the mouse model of ALI and pulmonary fibrosis with high successrate and low animal mortality, which provide an ideal experimental platform for further investigation.
Objective To explore a new method for the pre-degeneration of peripheral nerve in vitro for obtaining many effective Schwann cells so as to provide a large number of seed cells for the research and application of tissue engineered nerves. Methods The bone marrow derived cells (BMDCs) from transgenic green fluorescent protein C57BL/6 mouse and the sciatic nerve segments from the C57BL/6 mouse were co-cultured to prepare the pre-degeneration of sciatic nerve in vitro (experimental group, group A), and only sciatic nerve was cultured (control group, group B). At 7 days after culture, whether BMDCs can permeate into the sciatic nerve in vitro for pre-degeneration was observed by gross and immunohistofluorescence staining. And then Schwann cells were obtained from the sciatic nerves by enzymic digestion and cultured. The cell number was counted, and then the purity of primary Schwann cells was determined using immunohistofluorescence staining and flow cytometer analysis. Results At 7 days after pre-degeneration, gross observation showed that enlargement was observed at nerve stumps, and neuroma-like structure formed; the group A was more obvious than group B. Immunohistofluorescence staining showed many BMDCs permeated into the nerve segments, with positive F4/80 staining in group A. After culture, the yield of Schwann cells was (5.59 ± 0.19) × 104 /mg in group A and (3.20 ± 0.21) × 104/mg in group B, showing significant difference (t=2.14, P=0.03). At 48 hours after inoculation, the cells had blue bipolar or tripolar cell nuclei with small size and red soma by immunohistofluorescence staining; fibroblasts were flat polygonal with clear nucleus and nucleolus, showing negative p75NTR staining; and there were few of fibroblasts in group A. The purity of Schwann cells was 88.4% ± 5.8% in group A and 76.1% ± 3.7% in group B, showing significant difference (t=2.38, P=0.04). And the flow cytometer analysis showed that the purity was 89.6% in group A and 74.9% in group B. Conclusion BMDCs can promote the pre-degeneration of peripheral nerve in vitro, and it is a new method to effectively obtain Schwann cells for tissue engineered nerve.
Objective To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor α (TNF-α). Methods The primer of small interfering RNA (siRNA) coding sequence of silent TNF-α was designed and amplified, and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus, and the recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed. Then 64 female BABL/C mice (weighing, 20-25 g) were randomly divided into 4 groups (n=16): blank control (group A), positive control (group B), simple adenovirus (group C), and treatment group (group D). The prosthetic-model was established in group A, and the prosthetic-loosening-model in groups B, C, and D. At 2 weeks after modeling, PBS solution was injected first, and then the same solution was injected 24 hours later in group A; titanium particle solution was injected, and then PBS solution, Ad5 E1-CMVeGFP (1 × 109 PFU/mL), and Ad5-TNF-α-siRNA-CMVeGFP (1 × 109 PFU/mL) were injected, respectively in groups B, C, and D 24 hours later, every 2 weeks over a 10-week period. The general condition of mice was observed after operation. The tissues were harvested for histological observation, and the expression of TNF-α was detected by Western blot at 12 weeks after operation. Results The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector, and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP. All mice survived to the completion of the experiment. Histological observation showed that there were few inflammatory cells and osteoclasts in group A, with a good bone formation; there were a large number of inflammatory cells and osteoclasts in groups B and C, with obvious bone destruction; inflammatory cells and osteoclasts in group D was less than those in groups B and C, with no obvious bone destruction. Significant difference was found in the limiting membrane thickness and the number of osteoclasts (group A lt; group D lt; group B lt; group C, P lt; 0.05). Western blot showed that the TNF-α expression levels were 0.235 ± 0.022, 0.561 ± 0.031, 0.731 ± 0.037, and 0.329 ± 0.025 in groups A, B, C, and D respectively, showing significant difference among 4 groups (P lt; 0.05). Conclusion The recombinant adenovirus for silencing TNF-α is successfully constructed, which can effectively inhibit osteolysis by silencing TNF-α expression in the tissues around prosthesis in mice.