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Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.
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Cell Culture Techniques, Three Dimensional , Cell Culture Techniques , Apoptosis , Cell Proliferation , TechnologyABSTRACT
Objective:To evaluate the effect of edaravone on renal injury in rats with aldosterone-induced hypertension.Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-220 g, were divided into 3 groups ( n=8 each) using a random number table method: sham operation group (S group), hypertension group (H group) and edaravone group (E group). The hypertension model was developed by subcutaneously embedding aldosterone osmotic pump (administration rate 0.75 μg·kg -1·h -1) for 4 weeks.After embedding osmotic pump subcutaneously, edaravone 10 mg/kg was injected via the tail vein every day for 4 consecutive weeks in E group, while normal saline 10 ml/kg was injected instead for 4 consecutive weeks in H group.BP-2010A noninvasive manometry device was used to measure the systolic pressure of tail artery before embedding osmotic pump and at 1, 2, 3 and 4 weeks after administration.After 4 weeks of administration, the 24 h urinary albumin concentration, plasma creatinine (Cr) and blood urea nitrogen (BUN) concentrations were measured, the bilateral kidneys were weighed, the right kidney weight/body weight ratio (RKW/BW) was calculated, the glomerular extramesangial matrix area/glomerular area ratio (M/G) was measured by PAS method, and the collagen volume fraction (CVF) was measured by Masson staining method, and the expression of aldosterone receptor (MCR) and type Ⅰ collagen in renal tissues was detected by Western blot. Results:Compared with group S, the systolic pressure was significantly increased, the concentrations of 24-h urinary albumin, plasma Cr and BUN were increased, the RKW/BW ratio, M/G and CVF were increased, and the expression of type Ⅰ collagen and MCR was up-regulated after embedding osmotic pump in group H ( P<0.05). Compared with group H, the systolic pressure was significantly decreased, the concentrations of 24-h urinary albumin, plasma Cr and BUN were decreased, the RKW/BW ratio, M/G and CVF were decreased, and the expression of type Ⅰ collagen and MCR was down-regulated after embedding osmotic pump in group E ( P<0.05). Conclusions:Edaravone can reduce renal injury in rats with aldosterone-induced hypertension, and the mechanism is related to down-regulation of MCR expression.
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Objective:To determine the median effective dose (ED 50) of 0.5% ropivacaine when combined with dexmedetomidine based on femoral nerve cross-sectional area for ultrasound-guided femoral nerve block. Methods:American Society of Anesthesiologists physical statusⅠor Ⅱ patients of both sexes, aged 18-64 yr, with body mass index of 20-30 kg/m 2, scheduled for elective open reduction and internal fixation for patella fracture or removal of patella fracture by internal fixation, were randomly divided into dexmedetomidine and ropivacaine group (group DR) and ropivacaine group (group R). In group DR, 0.5% ropivacaine and 0.5 μg/kg dexmedetomidine were injected.In group R, 0.5% ropivacaine was injected.Ultrasonic localization of femoral nerve was performed for measurement of the femoral nerve cross-sectional area, and 0.5% ropivacaine was injected based on the area.ED 50 was determined by Dixon′ s up-and-down sequential method.The initial dose was 0.22 ml/mm 2, and the difference between the two successive doses was 0.02 ml/mm 2.The effective block was defined as complete loss of pain sensation in the areas of anterior skin of knee joint, skin on the inner side of the calf and dorsal medial skin of the foot and the degree of motor block was in stages 1-3 assessed using Brunnstrom motor function within 30 min after nerve block.Nerve block was considered ineffective if pain occurred in any nerve distribution area mentioned above.The study was terminated if 7 effective and ineffective alternating waves occurred.ED 50 and 95% confidence interval (CI) were calculated using Probit analysis. Results:In group R, 27 patients were enrolled in the study, and ED 50 (95%CI) of 0.5% ropivacaine for ultrasound-guided femoral nerve block was 0.106 (0.069-0.125) ml/mm 2.In group DR, 23 patients were enrolled in the study, and ED 50 (95% CI) of 0.5% ropivacaine for ultrasound-guided femoral nerve block was 0.038 (0.011-0.059) ml/mm 2.Compared with group R, ED 50 of 0.5% ropivacaine for femoral nerve block was significantly decreased in group R. Conclusion:When combined with dexmedetomidine 0.5 μg/kg, ED 50 of 0.5% ropivacaine based on femoral nerve cross-sectional area for ultrasound-guided femoral nerve block is 0.038 ml/mm 2.
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Objective:To determine the median effective dose (ED 50) of 0.5% ropivacaine based on femoral nerve cross-sectional area for ultrasound-guided femoral nerve block. Methods:Patients of both sexes, aged 18-64 yr, of American Society of Anesthesiologists physical status I or Ⅱ, with body mass index of 20-30 kg/m 2, scheduled for elective open reduction and internal fixation for patella fracture or removal of patella fracture by internal fixation, were enrolled in this study.Ultrasonic localization of femoral nerve was performed for measurement of the femoral nerve cross-sectional area, and 0.5% ropivacaine was injected based on the area.ED 50 was determined by Dixon′s up-and-down sequential method.The initial dose was 0.22 ml/mm 2, and the difference between the two successive doses was 0.02 ml/mm 2.The effective block was defined as complete loss of pain sensation in the areas of anterior skin of knee joint, skin on the inner side of the calf and dorsal medial skin of the foot and the degree of motor block was in stages 1-3 assessed using Brunnstrom motor function within 30 min after nerve block.Nerve block was considered ineffective if pain occurred in any nerve distribution area mentioned above.The study was terminated if 7 effective and ineffective alternating waves occurred.ED 50 and 95% confidence interval (CI) were calculated using Probit analysis. Results:Twenty-seven patients were enrolled in the study with the femoral nerve cross-sectional area (75±5) mm 2.ED 50 (95%CI) of 0.5% ropivacaine for ultrasound-guided femoral nerve block was 0.106 (0.069-0.125) ml/mm 2. Conclusion:ED 50 of 0.5% ropivacaine based on femoral nerve cross-sectional area for ultrasound-guided femoral nerve block is 0.106 ml/mm 2.
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Objective:To evaluate the relationship between edaravone-induced inhibition of pressure overload-induced myocardial remodeling and angiotensin Ⅱ type 1 receptor (AT1R)/mitogen activated protein kinases (MAPKs)/steroidogenic acute regulatory protein (StAR) signaling pathway in rats.Methods:Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 2 months, weighing 200-220 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (S group), pressure overload group (POL group) and edaravone group (E group). The cardiac pressure overload was induced by ligation of thoracic aorta for 8 weeks.After the model preparation, 0.9% sodium chloride 10 ml/kg was intraperitoneally injected daily in group POL, and edaravone 10 mg/kg was given instead in group E for 8 consecutive weeks.After the model was successfully established, the left ventricular ejection fraction (EF) and ventricular shortening fraction (FS) were measured by two-dimensional ultrasound.The animals were sacrificed by bloodletting, and the heart weight/body weight ratio (HW/BW ratio) was calculated.Myocardial tissues were obtained for determination of the cross-sectional area (MSA) after HE staining, the collagen volume fraction (CVF) (using Masson′s staining), the expression of AT1R and StAR (by immunohistochemistry), extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK phosphorylation levels (p-ERK1/2/ERK1/2 ratio and p-p38 MAPK/p38 MAPK ratio) (by Western blot) and the aldosterone content (by enzyme-linked immunosorbent assay). Results:Compared with group S, the HW/BW ratio, MSA and CVF were significantly increased, EF and FS were decreased, AT1R and StAR expression was up-regulated, and p-ERK1/2/ERK1/2 ratio, p-p38 MAPK/p38 MAPK ratio and aldosterone content were increased in group POL ( P<0.05). Compared with POL group, the HW/BW ratio, MSA and CVF were significantly decreased, EF and FS were increased, AT1R and StAR expression was down-regulated, and p-ERK1/2/ERK1/2 ratio, p-p38 MAPK/p38 MAPK ratio and aldosterone content were decreased in group E ( P<0.05). Conclusion:The mechanism of edaravone-induced inhibition of pressure overload-induced myocardial remodeling is probably associated with inhibiting the activation of AT1R/MAPKs/StAR signaling pathway in rats.
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Objective To evaluate the effect of propofol on high-mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4) signaling pathway during hepatic ischemia-reperfusion (I/R) injury in rats.Methods Thirty-six clean-grade healthy male Sprague-Dawley rats,aged 3 months,weighing 250 -300 g,were divided into 3 groups (n=12 each) using a random number table method:sham operation group (group S),hepatic I/R group (group I/R) and propofol group (group P).Hepatic I/R injury was induced by occluding the portal vein and hepatic artery supplying the left and middle lobes of the liver for 1 h followed by 6-h reperfusion in anesthetized rats.Propofol was infused via the tail vein at a rate of 12 mg ·kg-1 · h-1 starting from 20 min before ischemia until 6 h of reperfusion in group P.The rats were sacrificed at 6 h of reperfusion,and the left lobe of the liver was removed for microscopic examination of the pathological changes which were scored and for determination of the expression of HMGB1,TLR4,tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-6) in liver tissues (by Western blot).Results Compared with group S,pathological scores of liver tissues were significantly increased,and the expression of HMGB1,TLR4,TNF-α and IL-6 was up-regulated in I/R and P groups (P＜0.05).Compared with group I/R,pathological scores of liver tissues were significantly decreased,and the expression of HMGB1,TLR4,TNF-α and IL-6 was down-regulated in group P (P＜ 0.05).Conclusion The mechanism by which propofol reduces liver I/R injury is associated with blocking HMGB-1/TLR4 signaling pathway and inhibiting inflammatory responses in rats.
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Objective To evaluate the efficacy of Disposcope endoscope(DE)-guided nasotrache-al intubation in patients with difficult airway by comparing with fiberoptic bronchoscope(FOB). Methods One hundred and twenty American Society of Anesthesiologists physical statusⅠ-Ⅲ patients of both se-xes, with body mass index<25 kg∕m2, aged 18-64 yr, with mouth opening<3 cm, of Mallampati classifi-cation Ⅲ or Ⅳ, undergoing maxillofacial surgery requiring nasotracheal intubation were divided into DE group(n=60)and FOB group(n=60)using a random number table.Nasotracheal intubation was per-formed under the guidance of DE or FOB after induction of anesthesia.Glottis exposure was evaluated using Cormack-Lehane grade.Epistaxis during intubation, successful intubation, time and degree of glottis expo-sure, intubation time and development of tachycardia and hypertension and requirement for assisted ventila-tion with face mask during intubation were recorded.The patients were followed up postoperatively, and the development of intubation-related complications was also recorded.Results Compared with group FOB, glottis exposure time and incubation time were significantly shortened(P<0.05), and no significant change was found in Cormack-Lehane grade, rate of successful incubation, rate of successful intubation at first attempt or intubation-related complications in group DE(P>0.05). Hypertension and tachycardia were not found and no patients required assisted ventilation with face mask during intubation in the two groups.Conclusion DE-guided nasotracheal intubation provides similar efficacy to that of FOB with shorter time and is an optimal selection for the patients with difficult airway.
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Objective Getting the robust exogenous gene expression vector under the control of porcine insulin promoter, and to lay the foundation for pancreaticβ-cells specific transgene expressing pigs.Method Using porcine insu-lin promoter ( PIP, 1500 bp of the 5′UTR from the porcine INS gene including the first exon and the first intron) to con-struct expression vector, the HindIII restriction site which connected the sequences of PIP and EGFP was designed before ATG, named PIP-HindIII-EGFP.Considering that the different location of restriction site may affect the expression efficien-cy of the transgene, we optimized the expression vector.Firstly the HindIII restriction site was deleted to realize the seam-less connection of PIP and EGFP,the vector was named PIP-EGFP.Also we mutated the 3′intron splicing acceptor site( SA) of the first intron into HindIII restriction site, named as PIP-SA( M)-EGFP.Three different EGFP expression vectors were respectively transfected MIN-6 mouse pancreatic β-cells, pig ear fibroblasts and kidney cells.The transfected cells were cultured for 48 h and harvested for RT-PCR, flow cytometry and Western blot analysis, to analyze and compare the expres-sion efficiency of vectors.Results After transfection,green fluorescence was observed only in MIN-6 mouse pancreaticβ-cells.RT-PCR analysis and product sequencing showed that the three expression vectors did have different stability with in-tron splicing.The PIP-HindIII-EGFP construct and PIP-EGFP vector produced two kinds of mRNA with the first intron spliced and no spliced, indicating the instability of intron splicing.Mutation of the PIP splice site would cause the first in-tron not spliced, while flow cytometry and Western blot displayed that the mutation induced a most efficient expression of the downstream gene.Conclusions A robust and specific β-cells expression vector has been successfully generated by mutating the intron splicing acceptor site of the porcine insulin promoter.It provides the foundation for preparation of pigs with pancreaticβ-cells specifically expressing the transgene.
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Objective To investigate the effects of edaranvone on lung injury induced by myocardial ischemia-reperfusion (I/R) in rats. Methods Twenty-four male Wistar rats weighing 250-300 g were randomly assigned to one of 4 groups ( n = 6 each): group Ⅰ sham operation (group S); group Ⅱ myocardial I/R and group Ⅲ and Ⅳ different doses of edaravone ( group E1, E2 ). The animals were anesthetized, intubated and mechanically ventilated. In group Ⅱ-Ⅳ myocardial I/R was induced by occlusion of left anterior descending coronary artery for 45 min followed by 3 h reperfusion. In group Ⅲ and Ⅳ edavarone 3 and 10 mg/kg was administered via right femoral vein at 1 min before reperfusion respectively. The animals were sacrificed by exsanguination at the end of 3 h reperfusion. Blood was collected for determination of serum CK-MB activity and total protein content. The left lung was lavaged and the broncho-alveolar lavage fluid (BALF) was colleted for determination of protein content. Pulmonary permeability index (PPI) was calculated. The lung tissue was obtained for determination of BD-2 mRNA and protein and TNF-α expression. Results The serum CK-MB activity, PPI,BD-2 mRNA and protein and TNF-α expression were significantly higher in group I/R, E1 and E2 than in group S,but significantly lower in group E1 and E2 than in group I/R and in group E2 than in group E1. Conclusion Edaravone can reduce myocardial I/R-induced lung injury by scavenging oxygen free radicals and inhibiting inflammatory response of lung tissues in rats.