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Objective To clone express and purify Schistosoma japonicum fructose?1 6?bisphosphate aldolase SjFBPA in E. coli and observe its expression in different developmental stages of S. japonicum. Methods FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid and inducibly expressed with IPTG in E. coli BL21. SDS?PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA rSjFBPA . Then rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS?PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore SjFBPA mRNA was ana?lyzed in different developmental stages of S. japonicum by RT?PCR. Results SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro?tein could specifically reactive to the anti?His?tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT?PCR showed that SjFBPA mRNA was expressed in cercaria schistosomulum adult worm and egg of S. japonicum. Conclusion SjFBPA is successfully recombined and expressed in a prokaryotic system and SjFBPA mRNA is expressed in cercaria schistosomulum adult worm and egg of S. japonicum.
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Objective To observe if fluoxetine has a potency to inhibit the progression of Lewis lung cancer by combining the fluoxetine and cisplatin to treat the mice bearing Lewis lung cancer with depression .Methods We devel-oped a mouse model of Lewis lung cancer with depression which was intervened with cisplatin and fluoxetine , and the indi-cators related to cancer and depression were tested .Social interaction test was used to measure the behavioral changes of the depressed model mice .The serum cortisol and IL-6, BDNF mRNA in the hippocampus , and NF-κB and VEGF in the tumor tissue were selected for investigation and comparison .Results The mice which were induced by social defeat exhib-ited social avoidance behavior in the social interaction test .The cortisol and IL-6 level in both combination groups was de-creased compared with that in the model group (P0.05).Conclusions Fluoxetine has antidepres-sant effect by decreasing the high level of serum cortisol and promoting the BDNF mRNA expression in the hippocampus . However , fluoxetine is not found to have the potency to inhibit the expression of NF -κB related with the progression of tumor.
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Background Thyroid-associated ophthalmopathy (TAO) is a kind of clinically common and incurable ocular disease,and its incidence is at top place.The etiology and pathologic mechanism of TAO are still unknown because of shortness of replicative animal models and difficulty to acquire the ocular tissues in the early stage of the disease.To better understand the pathogenesis of TAO and investigate effective treatable measures, an appropriate animal model should be developed.Objective This study was to immunize female BALB/c mice with the recombinant plasmid of human thyroid-stimulating hormone receptor (TSHR) extracellular domain in cationic liposomes for the establishement of TAO models.Methods Thirty-two 6-to 8-week-old female BALB/c mice were randomly assigned to four groups according to computer random allocation.pcDNA3.1 +/hTSHR289 of 100 μg in an adjuvant cationic liposomes was injected via anterior tibialis muscle and peritoneal cavity separately in the recombinant plasmid injection group in 0, 3,6 weeks, and pcDNA3.1 or cationic liposomes was injected in the liposomes injection group or the blank plasmid group in the same way, respectively, and normal saline solution was injected in the blank control group.Body weight of the mice was measued before and 1 month,2,3 and 4 months after initial injection.The manifestations were observed after modeling.The mice were sacrificed 17 weeks after initial injection,and the histopathology examination was carried out on the thyroid gland and orbital tissue.The heart blood was collected from the mice,and serum contents of total thyroxin 4 (TT4) and thyroid-stimulating hormone (TSH)were assayed by ELISA.Results Protrusion, eyelid swell and keratitis occurred in 12 eyes of 6 mice in the recombinant plasmid injection group after immunization.A significant difference in the body weight of the mice was found among the blank control group, blank plasmid group, liposomes injection group and recombinant plasmid injection group (Fgroup =3.425, P =0.028), and the body weight was considerably reduced in the recombinant plasmid injection group in comparison with the blank control group, blank plasmid group,liposomes injection group (Ftime =0.838 ,P=0.023).The serum levels of TT4 were (7.75±1.00), (7.96±0.76), (6.76±1.10) and (4.43±2.88) μg/dlin the blank control group, liposomes injection group, blank plasmid group, and recombinant plasmid injection group, and those of TSH were (6.36±2.58),(4.83±3.96),(6.63±1.71) and (1.60 ±1.76) ng/ml, showing significant differences among the groups (F =7.150, P＜0.001;F =5.521, P＜0.01) , and the serum levels of TT4 and TSH were remarkably lower in the recombinant plasmid injection group than those of the blank control group,liposomes injection group and blank plasmid group (all at P ＜ 0.05).Histopathology revealed the lymphocyte infiltration of thyroid gland in 6 mice and proliferation of orbital adipose tissue, infiltration of lymphocytes and mastocytes,deposition of hyaluronic acid as well as swell, breakage and inflammatory cell infiltration of extraocular muscle in 15 eyes of the recombinant plasmid injection group.Conclusions A murine model of TAO can be successfully induced by immunization with recombination plasmid pcDNA3.1 +/hTSHR289 and cationic liposomes.The histopathology characteristics and ocular findings of the animal models are similar to human TAO.
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The cell-free plasma DNA of the 132 critically ill patients admitted to the emergency intensive care unit ( EICU) was measured by real-time quantitative polymerase chain reaction assay for the humanβ-globin gene. Ac-cording to the prognosis, the patients were divided into survival group (95 cases) and death group (37 cases). Blood samples were collected after EICU admission, 24 hours after admission, 48 hours after admission, and 1 week after admission. 30 age-mached controls were collected from healthy volunteers healthy care centre. Com-pared with control group, at each time point, cell-free plasma DNA concentration was significantly higher in EICU patients (P<0. 05). Cell-free plasma DNA concentrations were higher in hospital non-survivors than in survivors, of which plasma DNA concentration of 24 hours after admission and 48 hours after admission was statistically signifi-cant ( P<0 . 05 ) . The area under the ROC curves ( AUC ) for plasma DNA was bigger than the lactate and A-PACHE IIscore, the maximum of AUC was 0. 847(95% CI 0.648~1.000) at the point of 48 hours after admis-sion.
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<p><b>BACKGROUND</b>Bu-Shen-Yi-Qi-Tang (BSYQT), which is prescribed on the basis of clinical experience, is commonly used in clinics of traditional Chinese medicine (TCM) for asthma treatment. The components of BSYQT include Radix Astragali (RA), Herba Epimedii (HE) and Radix Rehmanniae (RR). The aim of this study was to compare the effect of granules and herbs of BSYQT on airway inflammation in asthmatic mice.</p><p><b>METHODS</b>Sixty female BALB/c mice were randomly divided into the normal control (NC) group, asthmatic group (A), decoction of granules of BSYQT treatment group (GD), decoction of herbs of BSYQT treatment group (HD), and dexamethasone treatment group (DEX). The mouse asthmatic model was induced by ovalbumin (OVA) sensitization and challenge. GD and HD of BSYQT as well as DEX were prepared and administered by intragastric infusion. Airway hyperresponsiveness (AHR) to methacholine (Mch), lung histopathology analysis, inflammatory mediators in serum (IL-4, IL-5, IL-17A, IFN-γ, and eotaxin) and in lung (IL-4, IL-5, IFN-γ, and eotaxin) were selected for investigation and comparison.</p><p><b>RESULTS</b>Both GD and HD treatment could decrease airway resistance (RL) and increase dynamic compliance (Cdyn) to Mch compared with the A group (P < 0.05). HD treatment was more effective in RL reduction than Mch at doses of 3.125 and 6.25 mg/ml (P < 0.05) and in Cdyn increase at Mch doses of 6.25 and 12.5 mg/ml (P < 0.05). There were no marked differences in RL reduction and Cdyn improvement between mice in HD and DEX groups (P > 0.05). Both GD and HD treatment markedly attenuated lung inflammation (P < 0.05), and HD treatment demonstrated more significant therapeutic function in alleviating lung inflammation than that of GD and DEX treatment (P < 0.05). Both GD and HD treatment resulted in a significant reduction in IL-4 and IL-17A levels and an increase in the IFN-γ level in serum compared with the A group (P < 0.05). The effect of HD in lowering the IL-4 and IL-17A level was significantly greater than that of GD (P < 0.05), and was not significantly different from DEX (P > 0.05). HD treatment significantly reduced the serum level of IL-5 and eotaxin compared with the A group (P < 0.05), however, mice in the GD treatment group did not demonstrate this effect. GD and HD treatment significantly reduced IL-4 and eotaxin mRNA expression compared with the A group (P < 0.05). HD treatment significantly reduced IL-5 mRNA expression compared with the A group (P < 0.05). There was a significant difference between the GD and HD treatment groups in reducing IL-5 and eotaxin mRNA expression (P < 0.05). HD treatment was more effective in down-regulation of IL-5 in serum and eotaxin level both in serum and lung than DEX (P < 0.05). Compared with the A group, an obvious increase in mRNA expression of IFN-γ was observed in both the GD and HD treatment groups (P < 0.05). However, the effect of HD treatment on increase of IFN-γ mRNA expression was more apparent than GD and DEX treatment (P < 0.05).</p><p><b>CONCLUSIONS</b>Both GD and HD treatment could decrease AHR, attenuate lung inflammation, reduce IL-4, IL-5, IL-17A, and eotaxin levels and increase IFN-γ levels in asthmatic mice. HD treatment manifests more remarkable inhibitory effects on asthmatic inflammation than GD treatment, which could provide a guide for further research on the screening of the material basis of the best anti-inflammatory effect of BSYQT.</p>
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Animals , Female , Mice , Asthma , Drug Therapy , Drugs, Chinese Herbal , Therapeutic Uses , Inflammation , Drug Therapy , Medicine, Chinese Traditional , Mice, Inbred BALB CABSTRACT
To probe the effect of paeoniflorin on periovular granuloma and liver fibrosis in mice infected with Schistosoma japonicum in different times of infection and the treatment with praziquantel (PZQ). The models of hepatic fibrosis induced by S.japonicum were established by exposure of BALB/c mice percutaneously through the tail to cercariae of S.japonicum. and mice with treatment were randomly divided into 3 groups: i.e. groups of pre-treatment (I), group of simultaneous treatment (Ⅱ) and group of post-treatment (III). All groups, except the normal control group, were orally introduced with PZQ. And mice in the paeoniflorin-treated group and control group were separately introduced with paeoniflorin and 0.5% sodium carboxymethycellulose respectively. The treatments in group I, II and III were started 30 days before PZQ usage, simultaneously with PZQ or 30days-after PZQ usage respectively. Mice in these groups were sacrificed on the 102, 132 or 162 days after infection. Then the serum levels of hyaluronic acid (HA), amino-terminal peptide of type III procollagen (PIIIP) and liver hydroxyproline (Hyp) were detected. The histopathology was examined by HE and Masson staining; the degree of hepatic fibrosis and the area of egg granuloma were analyzed. The expression of collagen I was examined by immunohistochemical method. It was found that the area of granuloma and degree of hepatic fibrosis in the paeoniflorin-treated groups in group I and III were significantly lower than those in the model control groups. Also, paeoniflorin could induce decreas expression of collagen I. Meanwhile the levels of serum HA, PIIIP and liver Hyp were all reduced in comparison with those in the control group (P<0.05 or P<0.01). However, in group Ⅱ, no significant difference was noted between the treated and the control group in most data. Paeoniflorin also showed the effects to reduce the size of periovular granuloma and to reduce the expression of type I collagen, thereby to resist the development of hepatic fibrosis caused by S. japonicum.-It is evident that PAE shows an efficaciously therapeutic effect on the development of liver fibrosis of shistosomiasis, whenever it is administered before or after the usage of schistosomicides.
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Background Thyroid-associated ophthalmopathy (TAO) is characterized by increased amount of orbital fat tissue.Leptin is a specific adipocytokine,and it may play an important role in the pathogenesis of TAO.Objective The present study investigates the effect of rh-leptin on the differentiation of orbital preadipocytes in subjects suffering from thyroid-associated ophthalmopathy (TAO).Methods The orbital fatty tissue was obtained from 7 patients with TAO during orbital decompression surgery.Orbital preadipocytes were cultured using the explant method and differentiated cells were identified with oil red staining.The subcultured preadipocytes were incubated with different concentrations of rh-leptin,and no rh-leptin was added in the control group.The average optical density of cytoplasm/nucleolus in differentiated adipocytes was measured by oil-red staining to evaluate the influence of different concentrations of rh-leptin on lipid formation of orbital adipocytes.Results Cultured preadipocytes in vitro were identified to be of mesenchymal origin by immunohistochemical staining.The average optical density of cytoplasm/nucleolus was 1.07±0.22,0.80±0.17,0.56±0.11,0.25±0.10,and 0.17±0.08,respectively in the control group,10μg/L of leptin group,50μg/L of leptin group,100μg/L of leptin group and 500μg/L of leptin group,showing a significant difference among the five groups (F=20.64,P=0.00).Compared to the control group,the lipid formation of orbital adipocytes gradually declined in various concentrations of rh-leptin (P＜0.05,P＜0.01).Conclusion Rh-leptin may suppress the differentiation and lipid formation of TAO orbital preadipocytes in vitro in a dose-dependent manner.Leptin may be a feedback modulator in TAO pathogenesis.
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To determine the inhibition of IL-13 by recombinant sIL-13Rα2 in NIH-3T3 fibroblast cells for its potential therapeutic value in hepatic fibrosis caused by Schistosoma japanicum in mice . IL-13 and sIL-13Rα2 from liver of BALB/c mice infected with S.japonicum at different infection time (weeks 0,6,8,10 and 12) were analyzed by ELISA and RT-PCR. The recombinant sIL-13Rα2 expression plasmidwas constructed, followed by transfection into NIH-3T3 fibroblast cells. TypeⅠcollagen produced by NIH-3T3 cells were examined by RT-PCR and Western blotting. It was demonstrated that the expression of IL-13 increased gradually after infection, reached peak density (16.1586 pg/mL)at week 8 and then reduced but was still higher than the level of control mice(3.4146 pg/mL;P =0.017 ). The secretion of sIL-13R α2 reached to its peak 10 weeks after infection(4827.426 pg/mL)and then reduced slowly but still higher than normal(4057.112 pg/mL; P=0.021). Meanwhile, the changes in mRNA level of IL-13 and sIL-13R α2 were coincided with that examined by ELISA. Both IL-13 and sIL-13Rα2 reached their peak density (P=0.033) at week 8 and 10 (P=0.025) respectively, and they were followed by a slower degree of decrease. The sIL-13Rα2 could significantly inhibit the effect of IL-13 on NIH-3T3 fibroblast cells, showing decreased mRNA level(P =0.012)and protein level of typeⅠcollagen compared with normal groups(P =0.031). It is concluded that the sIL-13Rα2 can inhibit the effect of IL-13 on NIH-3T3 fibroblast cells which leads to a reduced production of typeⅠcollagen, demonstrating its potential therapeutic value in hepatic fibrosis of schistosomiasis.
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Objective To observe the clinical and pathological characteristics of choroidal metastatic carcinoma from lung carcinoma. Methods The clinical and pathological data of 6 patients with choroidal metastatic carcinoma from lung carcinima were analysed retrospectively. Results All the 6 patients had severe visual impairment, including 3 with severe ophthalmalgia. Flat neoplasm were seen in the posterior pole of the eyes in all the 6 patients and retinal detachment were found in 5 patients. Fundus fluorescein angiography (FFA) examination had been performed on 1 patient and blocked fluorescence and hyperfluorescence were seen in the lesion with pinpoint fluorescein leakage loop around it. CT examination had been performed on 3 patients and the shadow of flat homogenous tumor was seen. MRI examination had been performed on 1 patient and high signal intensities on T1W and low signal intensities on T2W were found. In all the 6 patients with primary lung carcinoma, 5 were diagnosed with adenocarcinoma and 1 with cellule carcinoma through pathological examination, and 5 patients were diagnosed with choroidal metastatic carcinoma from adenocarcinoma and 1 with choroidal metastatic carcinoma from cellule carcinoma through pathological examination. Conclusion Rapid visual acuity decrease, severe ophthalmalgia, flat neoplasm in ocular fundus and secondary retinal detachment are the main clinical characteristics of the choroidal metastatic carcinoma from lung carcinoma. Most histopathological manifestations of the metastatic carcinoma like that of the primary focus, and adenocarninoma is the most common histoclassification.
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Objective To investigate the histopathological characteristics of choroidal melanoma. Methods The histopathological data from 64 patients with choroidal melanoma were analyzed retrospectively. The tumor size and the cytological types were observed and detected. The locations of the tumor were classified according to the involved part invaded by the anterior margine of the tumor, and the degrees of the development of the tumor were graded according to the extent of the outward infiltration of the tumor cells. Results In 64 patients with choroidal melanoma, There were large, medium, and small tumors in 25(39.1%), 31(48.4%), and 8(12.5%) respectively. The spindle cell type was found in 42 patients (65.6%) including spindle cell A and B type in 15(23.4%) and 27(42.3%) respectively; epithelioid and mixed cell type was found in 7(10.9%) and 10(15.6%) respectively; the other types were found in 5(7.8%). Twenty-five cases(39.1%)had no invasion with sclera, 22(34.4%)had but limited to sclera, 12(18.8%)penetrated through sclera and 5(7.8%)had intra-orbit infiltration. Conclusion The histopathological characteristics of choroidal melanoma are multiple, and spindle cell type is the most common one. The choroidal melanoma can easily invade the sclera.
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Objective To investigate clinical manifestation and pathologic changes of corneal incision rupture at different stages and the cause of incision rupture after abortive trauma.Methods Three removal eyeball obtained from RK for severe incision rupture by abortive trauma were fixed with formalin, embedded with paraffin, sected stained by HE and observed under light microscope.Results The eyeball distorted, intraocular structure disordered and vision lost severely in 3 eyes. Epithelial plugs were found in superficial layer of stroma and Bowman's layer broke and displaced in three cases. Healing and gap of incision presented 29 days after RK in 1 case. There were incision scar, collagen disarrangement and epithelial island in deep incision more than 2 years following RK in two cases, descent's layer broke in 1 case.Conclusion RK can weaken the eye which will rupture easily and their visual function will be disturbed severely after abortive trauma.
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Objective: To clone mouse rod opsin promoter (ROP) and establish transgenic mice harboring mouse rod opsin promoter and enhanced green fluorescent protein(mROP-EGFP) fusion gene. Methods: Mouse ROP was cloned from C57BL/6 mouse genomic DNA by polymerase chain reaction (PCR). Expression vector of mROP-EGFP fusion gene were constructed by recombination DNA technique. It was identified by restriction endonucleases digestion and confirmed by DNA sequencing. After Not I restriction endonuclease digestion, the coding elements were microinjected into male pronuclei of mice zygotes to generate transgenic mice. The pups were evaluated by PCR at genomic DNA level and mated with normal mouse. Expression of GFP in retina of transgenic mice was detected by fluorescent microscope. Results: 2. 1 kb mouse rod opsin promoter fragment was amplified from mice genome DNA. Expression vector pmROP-EGFP was constructed successfully. Following microinjection of coding sequence of pmROP-EGFP, 3 pups were verified to integrate the mROP-EGFP fusion gene in their genomic DNA by PCR assay, named C57-TgN (mROP-EGFP )SMMU21, C57-TgN (mROP-EGFP)SM-MU26 and C57-TgN(mROP-EGFP) SMMU27. They could express GFP in retina. Conclusion: 2. 1 kb mouse rod opsin promoter is cloned and expression vector pmROP-EGFP is constructed. mROP-EGFP fusion gene transgenic mice are established, which harboring mROP-EGFP gene and expressing GFP in their retina. This is valuable for studying the development of brain and retina, pathogenesis of retina disorder and retina transplanting.
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Objective To evaluate the effectiveness and safety of orbital decompression for compressive optic-neuropathy. Methods Fourteen eyes of twelve cases with Graves opthalmopathy and compressive optic-neuropathy undergone two orbital walls decompression with the follow-up period of more than 3 months were analyzed. Results The effect of complete closure of palpebral fissure was attained in all of the postoperative eyes and the visual acuity was increased in eleven eyes, remained no change in two eyes and decreased in one eye. The mean value of the recession of exophthalmic eyes after operation was mean 4 0 mm. Conclusion Two orbital walls decompression is an effective method for compressive optic-neuropathy in Gaves ophthalmopathy.
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Objective To explore the possibility of heterogenous gene to express in juvenile Schistosoma japonicum and the application of electroporation in transformation of schistosomulae. Methods The plasmids of pEGFP-C1 were introduced into mechanically transformed schsitosomula with electroporation. The presence, transcription and translation of the transgene in electroporated schistosomula were confirmed by PCR, RT-PCR and Western blotting analysis respectively using the genomic DNA, total RNA and protein extracted and isolated from schistosomula cultured in vitro for 48 hours. Meanwhile, localization of EGFP within electroporated schistosomula was performed with confocal laser scanning microscope. Results 760 bp and 276 bp amplified products by PCR and RT-PCR were found coincident with the expected size and expression of EGFP gene in elctroporated schistosomula was confirmed by Western blotting. Fluorescence of EGFP was localized in tegument and subtegument of the electroporated schistosomula with confocal microscopy, especially in the anterior part of the worm. Conclusion The heterogenous gene of EGFP has been successfully introduced into juvenile S. japonicum by electroporation and the expression of transgene was confirmed with molecular and microscopical methods.