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The T(EB4)Nta, T(IBj5)Nta, and T(B362i)NtA strains were constructed by introgressing the insertionaltranslocations EB4, IBj5, and B362i from Neurospora crassa into the related species N. tetrasperma. Theprogeny from crosses of T(IBj5)Nta and T(B362i)NtA with opposite mating-type derivatives of the standard N.tetrasperma strain 85 exhibited a unique and unprecedented transmission ratio distortion (TRD) that disfavoredhomokaryons produced following alternate segregation relative to those produced following adjacent-1 segregation. The TRD was not evident among the [mat A ? mat a] dikaryons produced following either segregation. Further, crosses of the T(IBj5)Nta and T(B362i)NtA strains with the Eight spore (E) mutant showed anunusual ascus phenotype called ‘max-4’. We propose that the TRD and the max-4 phenotype are manifestations of the same Bateson-Dobzhansky-Muller incompatibility (BDMI). Since the TRD selects against 2/3 ofthe homokaryotic progeny from each introgression cross, the BDMI would have enriched for the dikaryoticprogeny in the viable ascospores, and thus, paradoxically, facilitated the introgressions.
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Objective To establish and validate the Caco-2 cell in vitro absorption model,so as to play a foundation for next study of drug absorption and transport. Methods Caco-2 cells were cultured on the polycarbonate membrane inserts fixed in the 24-well transwell transport chamber with three types cell density of low,medium and high concentration (5×104,1×105,2× 105·mL-1) respectively.After being cultured for 3,6,9,12,15,18,21 d,the integrity of monolayer were assessed and compared by the cell morphology,growth characters and transepithelial electrical resistance (TEER),aiming to determine the inoculum density and culture time.Then the permeability and polarity were validated by the apical-to-basolateral amount of Lucifer Yellow across cell monolayer,the alkaline phosphatase activity in the apical side (AP),the basolateral side (BL) and intracellular activity. Results The cells of low,medium and high concentration group had fusion into a integrate cell monolayer and the maximum absorbance after being cultured for 15,12,9 d respectively.However,conglobated and dead cells were observed at the later growth stage in the medium and high concentration group and the TEER of cell monolayer were smaller than the low concentration group,which could reach 300 Ω·cm2after cultured 15 d and keep a relatively stable value, then cells were cultured with 5×104·mL-1cell density for 21 d.The Lucifer Yellow apparent permeability coefficient (Papp) was 3.57×10-7 cm·s-1which was lower than 5. 0×10-7cm·s-1according to provision. And the intracellular alkaline phosphates’ activity increased,AP/BL increased by 5 times in day 21. Conclusion The integrity,permeability and polarity of the established Caco-2 cell model in our laboratory was validated,and it can be used as an in vitro model similar with small intestinal epithelium for absorption and transport studies.
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Objective: To investigate the effect of borneol on puerarin through in vitro blood-brain barrier (BBB) model and to discuss the main pathway of borneol to promote the opening of BBB. Methods: MTT assay was conducted to investigate the toxic effects of borneol and puerarin with different concentration on the cells and to screen the concentration of tested drug. In vitro model of BBB was used to observe the effect of borneol on the opening of tight junction and the effect of borneol on puerarin through BBB. Results: The experimental drug concentration of borneol and puerarin was both 50 μmol/L by MTT experiment. There was no significant change in transepithelial electrical resistance (TEER) before administration and 24 h after administration, and the permeation rate of puerarin group and borneol + puerarin group were (59.96 ± 5.90)% and (106.80 ± 2.73)%, respectively, with significant difference between two groups. Conclusion: Borneol combined with puerarin can promote its permeation rate to a certain extent, but its mechanism needs to be further explored by cell-related tight junction proteins level and adenosine receptor signaling pathway.
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Objective Intestinal epithelial barrier damage is closely related to a variety of gastrointestinal disease,how to maintain its function effectively is the key to treat all these diseases.This research attempts to explore the protective effect and its mechanism of toll-like receptor 2 (Toll-like receptor,TLR2)on permeability of intestinal epithelial barrier by experiments in vitro,to lay a foundation for new treatment methods.Methods We cultured non-transfected Caco-2 cells,TLR2-deficiency Caco-2 cells,TLR2-overexpressed Caco-2 cells in normal control group until the 21 st d,then tested transepithelial electrical resistance(TEER) which reacts the permeability of epithelial barrier.We cultured 3 types of cells in inflammation group until the 19th d treated with 10 ng/ml IL-1 beta for 48 h,then tested TEER values at the 21st d.We treated 3 types of cells in inhibition group with PI3K/Akt pathway inhibitor for 1h befor IL-1 beta,then tested TEER values at the 21st d.Results TEER value of TLR2-deficiency Caco-2 cell monolayer significantly reduced (P ＜ 0.01),whereas TEER value of TLR2-overexpressed Caco-2 monolayer raised,but without statistically significant.TLR2 can prevent IL-1 beta caused TEER decreasing (P ＜ 0.01),but the effect disappeared after given PI3K/Akt pathway inhibitor.Conclusion TLR2 can regulate the permeability of intestinal epithelial barrier.In addition,TLR2 can protect permeability increasing caused by inflammation,this effect mediated by PI3 K/Akt pathway.
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Objective Inflammatory bowel disease is an important chronic gastrointestinal disease of childhood and adolescence.Intestinal mucosa barrier damage plays an important role in its pathogenesis.This study attempts to use IL-1β stimulating Caco-2 cell monolayer simulates inflammatory intestinal epithelial barrier in vitro,provides the basis for studying the pathogenesis and treatment of IBD.Methods Caco-2 cells were cultivated in vitro until the 21st day to simulate intestinal epithelial cell monolayer barrier.The cells of inflammation group one were disposed with IL-1β for 2 h since the 5th day and detected TEER every other day until the 21st day.The cells of inflammation group two were disposed with IL-1 β at the 18th day for 0,12,24,48,72h, and detected TEER respectively.Normal control group cells were cultured with common medium and detected TEER at the corresponding time point.Results The TEER of Caco-2 cells gradually increased from the 5th to 15th days,reached 600Ω·cm2 in the 15th day of a plateau until to the 21st day.Since the 5th day,the TEER of inflammation group one were all lower than normal group,and still to the 21st day ＜ 500Ω·cm2.Inflammation group two shows the time dependence TEER gradually reduce,peaked at 48 hours,then a slight increase in 72 hours.Conclusion The Caco-2 cells cultured for 2 ～ 3 weeks can form intestinal epithelial monolayer barrier with polarity,then treated with IL-1 β can manufacture inflammatory intestinal epithelial barrier model in vitro.
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Objective To establish and evaluate intestinal epithelial barrier model using Caco-2 cell so as to play a foundation for next study of barrier permeability.Methods Caco-2 cells were cultured in vitro then seeded into Transwell cell culture inserts.The permeability of the intestinal epithelial barrier was detected by transepithelial electrical resistance(TEER)and lucifer yellow flux,and verified by transmission electron micro-scope.Different concentrations of PAF(0,50,100,and 200 nmol /L)were exposed for 24 hours to Caco-2 mono-layer when cultured 21 days.The tight junction was observed under transmission electron microscope.Assess-ment of ZO-1 protein localization and expression were detected by immunofluorescence and Western blot analy-sis.Results Cultured Caco-2 cell confluencd as monolayer with time passed.From 5th day,TEER increased, then reached 600Ω?cm2 at 15th day and lasted to 21 st day,there was little flux of lucifer yellow,transmission e-lectron microscopy also found cells differentiated better,had well-arranged villi and polarity alined as monolayer, forming completed tight junction which was the marker of intestinal epithelial barrier model in vitro.TEER de-creased and lucifer yellow flux increased in cells exposed to PAF.The permeability reached the peak when ex-posed to 100 nmol /L PAF(P <0.01 ),tight junction disrupted,ZO-1 protein expression downregulated,abnor-mal localization and distribution was assessed by immunofluorescence staining.Conclusion Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.PAF increased intestinal epi-thelial permeability,which would correlate to the decreased protein expression and abnormal distribution of ZO-1.
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Objective To observe the influence of silent information regulator factor 1(SIRT1)on TNF-αinduced intestinal epi-thelial Caco-2 cell barrier function destroy and to investigate its molecular machenism.Methods Caco-2 cells were randomly divided into three groups:normal control group (control),TNF-αgroup (TNF-α,100 ng/mL for 24 h)and 100 ng/mL plus 40μm resvera-trol group (TNF-α+Res).Transepithelial electrical resistance (TER)was determined.SIRT1 and the protein expressions of ZO-1 , occludin were examined by using Western blot.Results The relative expression amounts of SIRT1 protein were 0.81 ± 0.02, 0.43±0.04 and 0.60±0.03 respectively.TER of three groups were (154.00±5.00),(97.00±4.00)and(128.00±6.00)Ohm/cm2 respectively.Compared with the control group,the expression of SIRT1 protein was reduced by 47% and TER was decreased by 37.00% in the TNF-αgroup.After resveratrol precondition,TER was increased by 32.00% compared with the TNF-αgroup. The relative expression amounts of ZO-1 and occludin protein in the control group,TNF-αgroup and TNF-α+Res group were (0.62±0.06,0.57±0.03),(0.23±0.05,0.33±0.04)and(0.41±0.03,0.50±0.02)respectively.After TNF-αtreatment,the ex-pressions of ZO-1 and occludin protein were significantly deceased(P<0.05),but the resveratrol precondition could attenuate this phenomenon,compared with TNF-αgroup,the protein expression was increased by 78.00% and 51.00% respectively (P<0.05). Conclusion Under the condition of TNF-αtreatment,the SIRT1 level is decreased,but increasing SIRT1 level could increase the in-testinal tight j unction protein ZO-1 and occludin protein expression,thus alleviate the damage of TNF-αon the epithelial barrier function constituted by Caco-2 cells.
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Objective To investigate the regulatory effects of hedgehog pathway on intestinal epithelial barrier function under hypoxia.Methods The IEC-6 cells of rats were divided into 3 groups:the normoxia group (21％ oxygen concentration),the hypoxia group (2％ oxygen concentration) and the hypoxia + cyclopamine group (cells pretreated by 5 mmol/L of cyclopamine,and then exposed in an atmosphere with 2％ oxygen concentration).The mRNA expressions of IHH,PTCH and GLI-1 were detected,and the transepithelial electrical resistance (TER) was determined.The protein expressions of tight junction proteins (ZO-1,Occludin,Claudin-1) and IHH were assayed by using the Western blot.All data were analyzed using the one-way analysis of variance or LSD-t test.Results The relative mRNA expressions of IHH,PTCH and GLI-1 were 0.056 ± 0.009,0.459 ± 0.087,0.142 ± 0.023 in the normoxia group,and 0.303 ± 0.052,0.678 ± 0.073,0.483 ± 0.061 in the hypoxia group,with significant difference between the 2 groups (t =-14.05,-11.85,-6.52,P ＜ 0.05).The relative protein expressions of IHH in the normoxia group and the hypoxia group were 0.39 ±0.06 and 0.91 ±0.15,with a significant difference between the 2 groups (t =-8.08,P ＜ 0.05).The TERs of the normoxia group,the hypoxia group and the hypoxia + cyclopamine group were (134 ± 5) Ohm/cm3,(100 ± 6) Ohm/cm3 and (118 ± 5) Ohm/cm3,with significant difference between the 3 groups (F =1.04,P ＜ 0.05).Compared with the normoxia group,the TER of the hypoxia group was decreased by 27.7％ (t =7.84,P ＜ 0.05) ; compared with the hypoxia group,the TER of the hypoxia + cyclopamine group were increased by 16.4％,but it was still significantly lower than the normoxia group (t =4.23,P ＜ 0.05).The expressions of ZO-1,Occludin and Claudin-1 were 1.18 ± 0.24,0.80 ±0.13 and 0.90 ±0.09 in the normoxia group,and 0.58 ±0.08,0.32 ±0.05 and 0.50 ±0.09 in the hypoxia group,and 0.92 ± 0.21,0.43 ± 0.10 and 0.82 ± 0.11 in the hypoxia + cyclopamine group,with significant difference between the 3 groups (F =4.95,2.88,10.09,P ＜0.05).The expressions of ZO-1,Occludin and Claudin-1 in hypoxia group were decreased by 48.7％,40.0％ and 55.6％ when compared with the normoxia group (t =12.86,9.35,18.90,P ＜0.05).The expressions of ZO-1,Occludin and Claudin-1 in the hypoxia + cyclopamine group were increased by 59.9％,35.2％ and 65.1％ when compared with the hypoxia group (t =5.63,2.92,6.66,P ＜ 0.05).Conclusion Hedgehog signal pathway could be activated under hypoxia,and then the expressions of tight junction proteins are decreased,which finally induces the injury of intestinal epithelial barrier function.