BACKGROUND: How to establish a stable in vitro culture system, including location of corneal limbal epithelial stem cells, in vitro sample harvest, in vitro culture, vector selection, as well as identification methods, play a key role in corneal limbal epithelial stem cells culture. OBJECTIVE: To culture the isolated rabbit corneal limbal epithelial stem cells and to identify the biological properties of cultured cells. METHODS: The primary rabbit cornel limbal epithelial stem cells were isolated and cultured with tissue inoculation using human amniotic membrane as vector. The growth features of cells were observed under an inverted microscope. The morphology of cells was observed by hematoxylin-eosin staining and a scanning electron microscope. Furthermore, the monoclonal antibody AE5 and P63 two-step immunohistochemical staining were used to identify limbal epithelial stem cell protein expression. RESULTS AND CONCLUSION: The rabbit corneal limbal epithelial stem cells could be successfully cultured and maintained a relatively high value-added potential in vitro. Rabbit corneal limbal epithelial stem cells cultured on the amniotic membrane pull netted cellular layer. The AE5 monoclonal antibody positive rate of primary cultured cells was about 5% and P63 monoclonal antibody positive up to 90%. AE5-positive rate increased and P63-positive rate decreased with the increase in the number of subculture. The rabbit limbal epithelial stem cells can be successful culture and amplified on human amniotic membrane in vitro by limbal tissue culture method. The cultured cells maintain the characteristics of corneal epithelial cells. The rabbit corneal limbal epithelial stem cells can form grafts on the amniotic membrane.

Objective To investigate the protective mechanism of Wenyang Fuyuan Prescription on nerve injury by improving brain iron metabolism in rats with cerebral ischemia-reperfusion injury(CIRI)based on ferroptosis.Methods A total of 72 SD rats were randomly divided into sham-operation group,CIRI model group,Wenyang Fuyuan Prescription group(18.0 g·kg-1,gavage),ferroptosis inducer group(100 mg·kg-1,intraperitoneal injection),Wenyang Fuyuan Prescription(18.0 g·kg-1,gavage)+ ferroptosis inducer group(intraperitoneal injection)and ferroptosis inhibitor group(5 mg·kg-1,intraperitoneal injection),12 rats in each group.All the procedures adopted in the sham group were the same as those in the model group.But nylon thread was inserted into the internal carotid artery at a depth of 9 mm and un-plugged middle cerebral artery.The rest of the groups were used to construct middle cerebral artery occlusion/reperfusion(MCAO/R)model by thread embolism method.Ferroptosis inducer(100 mg·kg-1)and ferroptosis inhibitor(5 mg·kg-1)were administered intraperitoneally to rats according to the grouping 24 hours before modeling.Wenyang Fuyuan Prescription(18.0 g·kg-1)was administered by gavage 2 hours after anesthesia and awakening.All intervention were given once daily for 7 consecutive days.The Longa scoring standard was used to evaluate the neurological deficit on 1,3,and 7 days after MCAO/R surgery,respectively.At the end of the treatment period,brain tissues were taken to observe the morphological changes of rat neurons in each group by hematoxylin eosin staining(HE).The ultrastructural changes of neuron mitochondria in each group were observed by transmission electron microscope.The biochemical kit was used to detect the content of iron ions(Fe2+)and reduced glutathione(GSH)in brain tissue.The protein and mRNA expressions of transferrin receptor 1(TFR1),iron regulatory protein 1(IRP1)and ferroportin(FPN)were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot.Results① Compared with sham group,the neurological deficit scores of rats in model group increased at each time point(P＜0.01).HE staining showed neurons were sparse and disordered,the nuclei underwent pyknosis,and vacuoles appeared at the edges.Under electron microscopy,it was observed that the number of neuronal mitochondria decreased,the density of mitochondrial membranes increased,massive numbers of mitochondrial membranes ruptured and dissolved,and mitochondrial cristae disappeared.The content of Fe2+,both mRNA and protein expressions of TFR1 were significantly increased(P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN were significantly decreased(P＜0.05,P＜0.01).② Compared with the model group,the neurological deficit scores of rats in the Wenyang Fuyuan Prescription group decreased at various time points(P＜0.05).The number of neurons increased,their arrangement was relatively neat,the morphology of the nucleus is complete and clear,the mitochondrial structure of neurons was relatively complete,the mitochondrial membrane was relatively intact,and the mitochondrial cristae were clear.The content of Fe2+,both mRNA and protein expressions of TFR1 were decreased(P＜0.05,P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN increased(P＜0.05,P＜0.01).③ Compared with the Wenyang Fuyuan Prescription group,the neurological deficit scores of rats in ferroptosis inducer group and the Wenyang Fuyuan Prescription + ferroptosis inducer group increased at all time points(P＜0.05).Distribution of neurons was in disorder,the nucleus shrinked,and vacuoles appeared at the edges.The density of mitochondrial membranes increased,some ruptured and dissolved mitochondrial membranes were found.The number of mitochondria decreased and mitochondrial cristae disappeared.The content of Fe2+,both TFR1 mRNA and protein expression increased(P＜0.05,P＜0.01),while the content of GSH,as well as expressions of mRNA and protein for IRP1 and FPN decreased(P＜0.05,P＜0.01).However,there was no statistically significant difference in all observed indicators between the ferroptosis inhibitor group and the Wenyang Fuyuan Prescription group(P＞0.05).Conclusion Wenyang Fuyuan Prescription can improve the neurological function and pathological damage of CIRI rats.Its mechanism may be related to regulating the expression of IRP1 protein,improving the brain iron metabolism pathway,and inhibiting ferroptosis.

Objective To explore the efficacy and safety of lung recruitment maneuver(LRM)on postoperative hypoxemia in patients with acute type A aortic dissection(ATAAD).Methods A total of 56 ATAAD patients with postoperative hypoxemia in the First Affiliated Hospital of Guangxi Medical University from November 2019 to May 2022 were selected and randomly divided into LRM group(n=36)and conventional treatment group(n=20).Patients in conventional treatment group received routine mechanical ventilation on the basis of lung protective ventilation.The patients in LRM group were treated with incremental positive end expiratory pressure(PEEP).Arterial blood gas analysis,respiratory parameters,hemodynamics parameters and serum interleukin(IL)-6 and IL-10 levels were compared between two groups before and after treatment.Results At 12h and 24h after treatment,arterial partial pressure of oxygen(PaO2),oxygenation index(OI),static compliance(Cstat)and dynamic compliance(Cdyn)in two groups were significantly higher than before treatment,the alveolar-arterial gradient of oxygen[PO2(A-a)],respiratory index(RI),peak inspiratory pressure(Ppeak)and plateau pressure(Pplat)were significantly lower than before treatment(P<0.05).PaO2,OI,Cstat and Cdyn in LRM group were significantly higher than those in conventional treatment group,PO2(A-a),RI,Ppeak and Pplat were significantly lower than those in conventional treatment group(P<0.05).Systolic blood pressure and mean arterial pressure decreased and central venous pressure increased during pulmonary reexpansion in LRM group(P<0.05),and all patients returned to baseline level after pulmonary reexpansion.At 12h after treatment,serum IL-6 and IL-10 levels in both groups were significantly lower than before treatment(P<0.05).Conclusion Incremental PEEP can improve oxygenation and lung compliance in patients with hypoxemia after ATAAD surgery,but it has transient effects on hemodynamics,and should be closely monitored during treatment.