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Objective To overcome the limitations of existing human respiratory syncytial virus(hRSV)animal models,such as semi-permissiveness and short duration of infection,this study established an IL2rg gene knockout(IL2rg-/-)rat model using TALEN gene editing technology.Methods The animal model was infected with hRSV intranasally.Clinical characteristics,body weight,and temperature changes were observed over the infection period(0～35 days).The total viral loads in respiratory organs,such as the nasal tissue,trachea,and lungs,were measured at various time points(4,11,20,and 35 days post-infection).Pathological analysis was conducted on target organs at the endpoint of observation(35 days post-infection).Changes in peripheral blood T,B,NK,and NKT cells and various cytokines were assessed at various time points(4,20,and 35 days post-infection).Results(1)IL2rg/-knockout rats sustained high viral loads in the nasal cavity upon intranasal inoculation with hRSV.The average peak titer rapidly reached 1 × 1010 copies/g in nasal tissue and 1 × 107 copies/g up to 5 weeks post-infection.(2)However,no significant pathological changes were noted in nasal,tracheal,or lung tissues.(3)An increase was observed in the content of peripheral blood B cells in hRSV-infected IL2rg--rats.(4)IL-6 and MCP-1 were increased in the early stage of infection and then decreased at the end of the observation period.Conclusions This study established a new IL2rg-/-rat model using TALEN technology and found that this model effectively supported high-level replication and long-term infection of hRSV,providing a good basis for antiviral drug screening and in vivo efficacy evaluation of anti-hRSV antibodies.
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ObjectiveThrough improving the potential of vascular endothelial growth factor receptor (VEGFR)-humanized mouse model (hKDR+/+) with C57BL/6N background to allow the growth of different mouse tumor cell lines, to establish novel tumor-bearing mouse models which can support in vivo tumorigenesis of different mouse tumor cell lines and be used to evaluate drugs targeting VEGFR.MethodsFirstly, a method to evaluate the in vivo activity of antibody targeting VEGFR based on the hKDR+/+ humanized mouse model was established. Recombinant activating gene 1 (Rag1) knockout mice (Rag1-/-) were established using CRISPR/Cas9 technology. Then these Rag1-/- mice were crossed with hKDR+/+ mice to get a double gene modified homozygous hKDR+/+/Rag1-/- mouse model by screening. Finally, tumor cell lines derived from different mouse strains were tested on the double gene-modified mouse model to compare the differences in tumor growth. ResultsAntibodies designed for VEGFR showed significant anti-tumor activity in hKDR+/+ mice, which significantly reduced tumor volume and weight compared with the PBS group (P<0.01, P<0.05). The number of B cells and T cells in the peripheral blood of Rag1-/- mice and hKDR+/+/Rag1-/- mice decreased (P<0.05, P<0.001). Tumors were observed in hKDR+/+/Rag1-/-, Rag1-/-, wild-type, and hKDR+/+ mice after 7 d of inoculation of MC38 cells derived from C57BL/6 mice. Tumors were only observed in groups of hKDR+/+/Rag1-/- and Rag1-/- mice, but not in the wild-type and hKDR+/+ mice after 10 d of inoculation with CT26 cells derived from BALB/c mice. After 3 weeks of inoculation, the tumor volume of hKDR+/+/Rag1-/- mice was significantly larger than that of Rag1-/- mice (P<0.01). ConclusionRag1 knockout mice were obtained and a novel hKDR+/+/Rag1-/- double genes modified mouse model was further screened. The tumor cell lines from different mouse strain origins were more prone to growth in mice with Rag1 gene deficiency. The results suggest that the reduced immune response of hKDR+/+ humanized mice will improve the capacity of supporting the growth of mouse tumor lines in the model. As a result, more tumor-bearing mouse models may be constructed for the evaluation of drugs targeting VEGFR in this way.
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Objective:To study the antimicrobial activity of diacerein on common pathogens of the ocular surface in vitro.Methods:Pathogens were collected from patients with ocular surface infections in Henan Eye Hospital, including Gram-positive cocci and bacilli, Gram-negative bacilli, filamentous fungi, and Candida.The antimicrobial activity of diacerein was determined by the K-B agar diffusion method, and its minimum inhibitory concentration (MIC) was determined by the micro-liquid method.Levofloxacin and voriconazole were used as the control of antibacterial and antifungal drug, respectirely.Results:Diacerein showed antibacterial activity against 42 strains of Gram-positive cocci and 10 strains of Gram-positive bacilli, its inhibition zone diameters for Staphylococcus epidermidis, S.aureus, S.intermedius and Gram-positive Corynebacterium were not significantly different from those of levofloxacin (all at P>0.05). Its MIC range of diacetate against Staphylococcus epidermidis, S. aureus, S. intermedius and other Staphylococci was 1-32 μg/ml, and its respective MIC 90 was 16, 8, 16, and 32 μg/ml.Diacerein had no bacteriostatic effect on 23 strains of Gram-negative bacilli, 10 strains of filamentous fungi and 3 strains of candida. Conclusions:Diacerein has antibacterial effects against Gram-positive Staphylococcus and Corynebacterium isolated from the ocular surface, but shows no antimicrobial activity against Gram-negative bacilli and fungi.Diacerein offers a new drug option and method for the treatment of bacterial keratitis.
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Objective@#To study the effects of platelets on macrophages phagocytosis and inhibition of fungi.@*Methods@#Macrophages were cultured, Fusarium Pythium spores were extracted and platelets were isolated from blood of mouse.Simple spore group, spore+ macrophage group and mixed platelet group were set, and were inoculated with fungal spore, equal proportion spore+ macrophage and platelet+ spore+ macrophage, respectively.The prepared plate was placed on a spinning disk laser scanning confocal microscope at 1 hour, 2, 3 and 4 hours after culture, five visual fields were randomly selected at the corresponding time points for photography.The phagocytic rate, phagocytic index and fungal spore germination rate were calculated.Fungal hyphae length of each group at 4, 6 and 8 hours after culture were calculated.The single macrophage group, spore+ macrophage group and mixed platelet group were set and the cytotoxicity was measured by real-time cell analyzer.The breeding and use of mice was in according with the ARVO statement.This study was approved by the Experimental Animal Ethics Committee of Henan Eye Institute (HNEECA-2017-04).@*Results@#The phagocytic rate and phagocytic index of macrophages in mixed platelet group at 1 hour, 2, 3 and 4 hours after culture were significantly higher than those in spore+ macrophage group at corresponding time point (all at P<0.05). There were significant differences in spore germination rate at 1 hour, 2, 3 and 4 hours after culture among different groups (H=60.05, 37.89, 55.15, 60.52; all at P<0.001). The spore germination rates of spore+ macrophage group at 1 hour, 2, 3 and 4 hours after culture were lower than those of simple spore group, while the spore germination rates of mixed platelet group at 1 hour and 3, 4 hours after culture were lower than that of spore+ macrophage group, the differences were statistically significant (all at P<0.01). There were significant differences in fungal hyphae length at 4, 6 and 8 hours after culture among the three groups (H=13.76, 43.57, 60.87; all at P≤0.001). The fungal hyphae lengths of spore+ macrophage group at 4, 6 and 8 hours after culture were lower than those of simple spore group, and the fungal hyphae lengths of mixed platelet group at 6 and 8 hours after culture were lower than those of spore+ macrophage group at the corresponding time point.The differences were statistically significant (all at P<0.05). There was no significant difference in cell index between 0 hour, 6, 12, 18 and 24 hours after culture (F=0.02, 1.08, 1.61, 1.58, 2.52; all at P>0.05). There were significant differences in cell index among different groups at 30, 36, 42 and 48 hours after culture (F=10.81, 8.08, 5.61, 5.72; all at P<0.05). The cell indexes in spore+ macrophage group at different time points were significantly lower than those in simple macrophage group (all at P<0.01).@*Conclusions@#Platelets can promote the phagocytosis and inhibition of macrophages on fungi, and platelets may have antagonistic effect on fungal cytotoxicity.
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Objective This study was to assess the efficacy of corneal collagen cross-linking treatment on fungal keratitis.Methods Eighty SPF male C57BL/6 mice aged 6-8 weeks were selected for the experiment.Fusarium solani infected model was established on the left eyes of all 80 mice.Forty mice were distributed randomly into sham operation group,model control group,scraped epithelium group and corneal collagen cross-linking (CXL)group (treated with epithelium scraped and CXL).Three days after modeling,the levels of the corncal disease sevcrity were scored by slit lamp microscopy.The fungal activity was confirmed by plate counts.The left 40 mice were divided randomly into sham operation group,model control group,scraped epithelium group and CXL group (treated with epithelium scraped and CXL).In 1 day and 2,3,4,5,6,7,14 days after modeling,the corneas were examined under the slit lamp microscope.The corneal pathological examination of each group were conducted with hematoxylin and eosin staining at postoperative 14 days.The animal feeding and use was in accordance with the standards set by the ARVO,and the experiment was approved by the Ethic Committee for Experimental Animal of Henan Eye Institute.Results The colony-forming units (CFUs) of fungal solutions in culture significantly decreased with CXL treatment (F =11.97,P =0.00).The Pearson correlation analysis of CFU and clinical scores in CXL group showed that inflammatory cells infiltration was positively correlated with corneal disease severity (r =0.723,P =0.043).Corneal inflammatory score was significantly lower in the CXL group in various time points,with a significant differences among the groups and time points (Fgroup =34.44,P=0.00;Ftime =17.49,P=0.00).Corneal lesion and the depth of ulceration in scraped epithelium group and CXL group were remarkably lower than that in the model control group (all at P ＜ 0.05).Histopathology revealed that the degree of corneal collagen fibers destruction and the ratio of inflammatory cells infiltration in scraped epithelium group (59.33％) and CXL group (11.29％) were much lower than that in the model control group (73.65％).Conclusions CXL can inhibit the fungal activity effectively in the cornea of mice,and reduce the fungal induced keratitis reaction.
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Objective To investigate the effect of MMP-8 on cornea. Methods Fifteen C57BL/6J healthy mice were selected. The right eyes corneal stroma was injected by 10μL MMP-8 as the experimental group and the left eyes were injected by same amount of normal saline as the control group. At 0,4,8 h, the two-photon microscope second harmonic generation imaging technology was used to scan mice corneal stroma layer by layer in vivo. The obtained images were performed the 3D reconstruction by Imaris software and the signal intensity of the images were calculated. At 4,8 h, the corneal opacity degree was evaluated under slit lamp. At 8 h,mice were killed and corneas were collected to determine the hydroxyproline concentration. Results The cornea stromal fiber signal strengthes at 0 h in the experimental group and control group were (89.7±11.2) and (85.3±7.0),which at 4 h were (14.5±3.4) and (46.6±14. 0) respectively,which at 8 h were (11.0±4.6) and (34.6±12.5) respectively. The cornea stromal signal strength at 4,8 h in the experiemental group was significantly decreased compared with that in the control group (P<0.05) ;the cornea at 4 ,8 h in the experimental group was significantly turbid than that in the control group (P<0.05);the cornea hydroxyproline concentrations detected at 8h in the experiemental group and control group were (0.433±0. 090) μg/mg and (0. 590±0. 133) μg/mg respectively,the experimental group was significantly lower than the control group (F=7. 193,P=0. 014). Conclusion MMP-8 has obvious degradation and destroy effect on mice corneal stroma collagen,which leads to the decrease of corneal opacity.
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Objective To obtain the basic growth parameters of a self-established F1 hybrid CB6F1 C57-ras transgenic mouse model, and to provide basic information for commercialization of this mouse model. Methods F1 hybrid mice (CB6F1) were produced by crossing C57-ras heterozygous transgenic (c-Ha-ras+/-) male mice and wild-type BALB/cJ female mice.The average litter size, weaning rate, sex ratio, growth performance and C57-ras transgenic positive rate were recorded and analyzed.Results The average litter size was eight, weaning rate was 90%, and sex ratio was approximately in accordance with prediction.The average body weight of newborn mice was 1.73 ±0.05 g.The average body weight of 10-week old c-Ha-ras transgenic female and male mice in CB6F1 background was 24.38 ±1.74 g and 29.42 ±1.72g, respectively, which had a significant difference (P<0.01).The c-Ha-ras transgenic positive rate was 46.9%. which was in accordance with genetic rules.Conclusions The F1 hybrid mice (CB6F1) produced by crossing C57-ras heterozygous transgenic ( c-Ha-ras +/-) male mice and wild-type BALB/cJ female mice show normal growth performance and development characteristics, and it can be used for large-scale commercial supply.
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Objective To determine the normal range of hematological and visceral weight parameters of F 1 4-week and 8-week old, male and female transgenic CB6F1 mice.The influence of gender and week age on the biochemical parameters was assessed .Methods 4-week and 8-week old CB6F1 mice, half male and half female ( n=20 in each group) , were weighed alive , dissected to weigh the main viscera , and blood samples were collected to test the physiological and biochemical parameters .Results When 4-week old and 8-week old CB6F1 mice were compared , there were significant differences in 22 parameters (body weight, heart, liver, spleen, left ovary, left testis, right testis, WBC, RBC, HGB, HCT, MCV, MCH, PCT, MPV, PDW, LYM, TP, ALT, ALB, P and TG) (P<0.01 for all), and in 8 parameters (left kidney, right kidney, right adrenal, thymus, left ovary, RDW, MON%and BUN) (P<0.05 for all). When male and female 4-week CB6F1 mice were compared, there were significant differences in 14 parameters ( body weight, heart, liver, spleen, lung, left kidney, right kidney, MCHC, LYM, ALT, ALP, GLU, P and CHO) (P<0.01 for all), and in 6 parameters (right adrenal, WBC, PCT, MPV, TP and BUN) (P<0.05 for all).For male and female 8-week old CB6F1 mice, there were significant differences in 15 parameters (body weight, heart, liver, lung, left kidney, right kidney, MCV, PCT, LYM, LYM%, NEUT%, ALT, GLU, P and CHO) (P<0.01 for all), and in 5 parameters (WBC, RBC, MPV, NEUT and TP) (P<0.05 for all).Conclusions The normal range of hematological and visceral weight parameters of 4-week and 8-week old male and female CB6F1 mice are determined.Our study establishes normal detection indexes of CB6F1 mice and provides useful reference for its application .
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Objective To improve the gene targeting efficiency with C57BL/6 embryonic stem ( ES) cells.Meth-ods Three different genetically modified C57BL/6 ES cell lines, named TLX3, Ai3K and SL, were microinjected into ICR, B6( Cg)-Tyrc-2J and BALB/c mouse blastocysts, respectively.The efficiency was statistically evaluated according to three aspects:blastocyst collection, chimera production and germline transmission.Results None of the three ES cell lines was germline transmitted with B6(Cg)-Tyrc-2J mice as blastocyst donors, while it was achieved with both BALB/c and ICR mouse blastocysts.Compared in the aspect of blastocysts collection, ICR mouse was much better than BALB/c mouse (P<0.05), and the chimera production efficiency of ICR mouse was comparable to that of BALB/c mouse (P =0.115). As to the germline transmission efficiency, that of BALB/c mice is significantly higher than that of the ICR mice ( P<0.01).Conclusions The germline transmission efficiency of BALB/c mouse is highest among these three mouse strains. However, it has the disadvantages of blastocyst collection, developmental delay and zona pellucida fragility, compared with ICR mouse.Therefore, ICR mouse is also a good candidate as blastocyst donor for embryonic stem cell microinjection.
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Background Nanoemulsions (NEs) is one of the most popular ophthalmic colloidal drug delivery system due to its long-term stability, low toxicity and irritancy, considerable capacity for solubilization of lipophilic drug molecules and great potential in bioavailability improvement.The cornea pathway is the main route of intraocular absorption after topical use of NEs.Though NEs possess numerous physiological and physicochemical advantages,the use of NEs cannot always obtain satisfactory results.Objective This study was to investigate the impacts of epithelium and stroma on the corneal permeation of topical ophthalmic terbinafine hydrochloride nanoemulsions (TH-NEs).Methods TH-NEs was prepared by the self-emulsification method.The size and Zeta potential of the oil droplets in the formulation were analyzed using a dynamic light-scattering particle size analyzer.The high performance liquid chromatography (HPLC) was used for the in vitro release study.Sixty New Zealand albino rabbits were randomly divided into intact cornea group and cornea epithelium debrided group.The cornea epithelium of the left eyes was debrided in the cornea epithelium debrided group.The TH-NEs were instilled into the lower conjunctival sac of left eyes.Six rabbits were executed from each group 15,30,60,120 and 240 minutes after dosing,respectively.The corneas were collected and analyzed by HPLC.The fluorescein diacetate (FDA) was used to label the TH-NEs.Two C57BL/6 mice with left cornea epithelium debrided and 2 normal mice were used for the fluorescence tracing study.The fluorescence distribution of FDA labeled TH-NEs was observed by a two-photon laser confocal scanning microscope 30 minutes and 60 minutes after single instillation.Results The average size and Zeta potential of the oil droplets were 51.37 nm and-0.232 7 mV respectively,and 0.482％ of encapsulated drugs was released from the TH-NEs after 12 hours.The peak concentrations of TH in the intact cornea and epithelium debrided cornea were (17.85 ± 2.79) μg/g and (4.40± 1.75) μg/g respectively, which occurred 15 minutes postdose.The drug concentrations in the intact cornea were significantly higher than that in the debrided cornea 15,30,60 and 120 minutes after dosing, with significant differences between them (t =9.998,8.658,6.903,7.576;all at P=0.000).The fluorescence was observed in the cornea epithelium when the cornea was intact.The fluorescence intensity in the superior layer of corneal epithelium was obviously higher than that in the deep layers of corneal epithelium 30 minutes and 60 minutes after dosing.No fluorescence was observed in the cornea stroma of both eyes.Conclusions The cornea epithelium is the main of absorption and distribution position of TH-NEs.The cornea stroma is the dominating permeation barrier for the intraocular transportation of the TH-NEs.The cornea stroma may stop the permeation of TH-NEs by molecular exclusion mechanism.
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Objective To establish a simple , fast and economic total DNA extraction method to serve the rapid identification of model mouse genotype in large number of mice .Methods Three methods, i.e.phenol extraction, isopropyl alcohol precipitation and mouse ear boiling methods were used to extract the total DNA from ten C 57-rasmodel mice.The purity and yield of DNA obtained by the three methods were compared , and polymerase chain reaction ( PCR) assay was used to compare the efficacy of the three extraction methods .Results Among the three methods , phenol extraction was the best and isopropyl alcohol precipitation was the poorest in DNA yield .In terms of DNA purity , the phenol extraction was the best and the mouse ear boiling method was the poorest .All the three methods could be used to extract the total DNA from mice serving as template of PCR reaction for the mouse genotype identification .The time consumption of three methods are 12.5 hr ,13 hr and 0.18 hr.Mouse ear boiling method was significantly lower than that of the other two methods ( P <0.01 ) ,.The obtained total DNA can be stored at conventional -20℃for 7 days and 30 days later still can be used as a template for PCR reaction .Conclusions Among the three methods studied , the mouse ear boiling method is simple and with the lowest cost , so it is feasible for total DNA extraction in scaled genotyping experiments .